Polypeptides

ABSTRACT

There is provided inter alia a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR1-CDR3 and FR1-FR4 are as defined in the specification.

CROSS REFERENCE

This application is a continuation of international application PCT/EP2017/051237 filed on Jan. 20, 2017 which derives priority from EP 16152321.2 filed on Jan. 21, 2016 and EP 16191988.1 filed on Sep. 30, 2016, the contents of each of which are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to polypeptides comprising an immunoglobulin chain variable domain (or ‘ICVD’) which binds to the interleukin-6 receptor (IL-6R) as well as to constructs and pharmaceutical compositions comprising these polypeptides. The present invention also relates to nucleic acids encoding such polypeptides, to methods for preparing such polypeptides, to cDNA and vectors comprising nucleic acids encoding such polypeptides, to host cells expressing or capable of expressing such polypeptides and to uses of such polypeptides, pharmaceutical compositions or constructs.

BACKGROUND OF THE INVENTION

IL-6 induces cell activation via a receptor system that consists of two receptor chains: a ligand-specific non-signalling transmembrane IL-6 receptor alpha subunit (the membrane-bound form of IL-6R, also known as mIL-6R, IL-6Rα, gp80 and CD126) and a second trans-membrane receptor chain gp130 that is required for signal transduction. In classical (cis) IL-6 signalling, IL-6 first binds to the membrane IL-6Rα subunit, which in turn associates with gp130 to form an IL-6-receptor complex that is able to induce cell activation. The restricted expression of membrane IL-6Rα receptors limits this classical IL-6 signalling mechanism to a few cell types including hepatocytes, neutrophils, monocyte/macrophages, some lymphocyte subtypes and intestinal epithelial cells. A second form of the IL-6R comprising the extracellular ligand-binding region of the IL-6Rα-subunit has also been identified. The soluble form of the IL-6R (sIL-6R) is generated by protease mediated shedding of IL-6Rα ecto-domains from membrane IL-6R expressing cells or is secreted from the cells as an alternatively spliced product. Importantly, IL-6 can still bind to the sIL-6R and the IL-6/sIL-6R complexes formed can associate with gp130-receptor chains to induce signalling. As a wide range of cells express gp130 but not the IL-6R, this process termed “trans-signalling” provides a mechanism for extending the range of cell types that are capable of responding to IL-6 and this process appears to be particularly important for the development and perpetuation of chronic inflammation (Rose-John 2012).

Inhibition of the IL-6R has potential for therapeutic benefit in autoimmune diseases such as Crohn's disease (CD) and ulcerative colitis (UC). Systemically administered IL-6 pathway antagonists including both IL-6 and IL-6R blocking antibodies have demonstrated efficacy without major toxicity in inflammatory diseases including rheumatoid arthritis and Castleman's disease. Tocilizumab, a humanised IL-6R monoclonal antibody that targets both membrane and soluble IL-6Rs (cis/trans signalling inhibitor) has also shown evidence of clinical efficacy in a pilot clinical study in patients with Crohn's disease (Ito et al., 2004).

CD and UC are diseases of the gastrointestinal tract in which excessive production of IL-6 and the release of sIL-6R are both localised within inflamed mucosal and sub-mucosal intestinal tissues. Preclinical studies have shown that the production of IL-6 in ex vivo cultures of inflamed IBD tissue greatly exceeds the release of sIL-6Rs. The ability to deliver an oral therapeutic agent with exposure and IL-6R antagonist activity limited to the gut offers potential for efficacy similar to (or greater than) tocilizumab, but with potentially improved safety due to the reduced systemic exposure. Unlike tocilizumab (or other systemically administered anti-IL-6R targeted antibodies), an orally delivered IL-6R antagonist would not be required to neutralise the large pool of sIL-6R that is present in the circulation in addition to the neutralisation of tissue sIL-6R production.

In inflamed IBD tissue, the production of IL-6 and shedding of IL-6Rs from activated macrophages results in the formation of soluble IL-6/sIL-6R complexes that can activate trans-signalling in cells that express only the IL-6R gp130 subunit. This mechanism, which extends IL-6 responsiveness to an increased number of target cells, is considered to play an important role in the orchestration of mucosal inflammatory processes. The mechanism of IL-6 induced intestinal epithelial cell proliferation and regeneration is thought to involve signal transduction mediated via membrane bound IL-6 receptors (cis-signalling) (Rose-John, 2012). The anti-human IL-6R antibody tocilizumab blocks both IL-6R classic signalling and IL-6/sIL-6R mediated trans-signalling and therefore blocks both pro-inflammatory and potentially protective activities of IL-6. A rationale for the development of selective antagonists of IL6-trans-signalling has been proposed based on the concept that this might avoid the inhibition of potential beneficial epithelial regenerative effects of IL-6 (see Rose-John, 2012; Waetzig et al., 2012) that are mediated via mIL-6Rs (cis-signalling). An oral IL-6R antagonist with this profile could have safety and efficacy advantages over existing IL-6 neutralising antibodies for the treatment of Crohn's disease based on anti-inflammatory and improved mucosal healing properties.

WO2008020079, WO2008071685, WO2009095489, WO2010115995, WO2010115998 and WO2013041722 (herein incorporated by reference in their entirety) disclose single domain antibodies directed against IL-6R and related aspects.

Polypeptides of the present invention may, in at least some embodiments, have one or more of the following advantages compared to anti-IL-6R substances of the prior art:

-   -   (i) increased affinity for IL-6R;     -   (ii) increased specificity for IL-6R;     -   (iii) increased neutralising capability against IL-6R;     -   (iv) increased cross-reactivity with IL-6R from different         species such as human and cynomolgus monkey;     -   (v) reduced immunogenicity, for example when administered to a         mouse, cynomolgus monkey or human;     -   (vi) increased stability in the presence of proteases, for         example (a) in the presence of proteases found in the small         and/or large intestine and/or IBD inflammatory proteases, for         example trypsin, chymotrypsin, MMP3, MMP10. MMP12, other MMPs         and cathepsin and/or (b) in the presence of proteases from gut         commensal microflora and/or pathogenic bacteria, actively         secreted and/or released by lysis of microbial cells found in         the small and/or large intestine; (vii) increased stability to         protease degradation during production (for example resistance         to yeast proteases);     -   (viii) increased suitability for oral administration;     -   (ix) increased suitability for local delivery to the intestinal         tract and lamina propria following oral administration;     -   (x) increased suitability for expression, in a heterologous host         such as bacteria such as Escherichia coli, or a yeast or mould         (e.g. those belonging to the genera Aspergillus. Saccharomyces,         Kluyveromyces, Hansenula or Pichia, such as Saccharomyces         cerevisiae or Pichia pastoris);     -   (xi) suitability for, and improved properties for, use in a         pharmaceutical;     -   (xii) suitability for, and improved properties for, use in a         functional food;     -   (xiii) improved tissue penetration such as penetration of         inflamed colonic mucosal epithelium and submucosal tissues to         access the sub mucosal lamina propria;     -   (xiv) increased suitability for formatting in a multispecific         format;     -   (xv) increased selectivity for inhibition of IL-6R         trans-signalling over cis-signalling;     -   (xvi) binding to novel epitopes.

Advantages (i) to (xvi) above may potentially be realised by the polypeptides of the present invention in a monovalent format or in a multivalent format such as a bihead format (for example homobihead or heterobihead formats).

SUMMARY OF THE INVENTION

The present inventors have produced surprisingly advantageous polypeptides comprising immunoglobulin chain variable domains which bind to IL-6R. These polypeptides in particular benefit from surprisingly high potency. In some embodiments, they are also capable of cross-reacting with cynomolgus monkey IL-6R and in some embodiments, remain stable on exposure to proteases of the small and large intestine. In one embodiment, these polypeptides have undergone further enhancement by engineering. These further enhanced polypeptides benefit from the above advantages, retain their IL-6R-neutralising activity during passage through the intestinal tract and further resist degradation and/or inactivation by proteases of the intestinal tract, for example, digestive, inflammatory and microbial proteases from, for example, multiple mammalian species (rodent, pig, non-human primate and human).

It may be expected that these polypeptides have particular utility in the prevention or treatment of autoimmune and or inflammatory disease such as inflammatory bowel disease (for example Crohn's disease or ulcerative colitis), or in the prevention or treatment of mucositis, particularly when administered orally.

The present invention provides a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 75% or greater sequence identity with SEQ ID NO: 3.

The present invention also provides a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from V33, G52, G56 and Y93, and optionally one or more amino acids selected from T18, T21 and F62, according to Kabat numbering.

DESCRIPTION OF THE FIGURES

FIG. 1—Inhibition of IL-6/IL-6R/gp130 interaction by ICVDs (3C12, tocilizumab, 7F6, 4D3 and 5G9)

FIG. 2—Inhibition of IL-6/IL-6R/gp130 interaction by ICVDs (7F6 and 21E6)

FIG. 3—Inhibition of gp130/IL-6/IL-6R interaction by ICVDs (3C12, 4D3, 7F6, 5G9, 20A11 and tocilizumab)

FIG. 4—Inhibition of human gp130/human IL-6/cynomolgous monkey IL6-R interaction by ICVDs (3C12, 4D3, 7F6, 5G9, 20A11 and tocilizumab)

FIG. 5—Inhibition of IL-6-dependent proliferation of TF-1 cells (7F6, 5G9, 4D3, 3C12, tocilizumab Fab and 20A11)

FIG. 6—Inhibition of IL-6-dependent proliferation of TF-1 cells (7F6 and 21E6)

FIG. 7—Phosphoprotein signals for UC2045, 4 hour incubation (ID-123V)

FIG. 8—Phosphoprotein signals for CD2052, 4 hour incubation (ID-123V)

FIG. 9—Phosphoprotein signals for CD2059, 4 hour incubation (ID-123V)

FIG. 10—Phosphoprotein signals for CD2061, 4 hour incubation (ID-123V)

FIG. 11—Phosphoprotein signals for UC2075, 4 hour incubation (ID-123V)

FIG. 12—Phosphoprotein signals for UC2045, 24 hour incubation (ID-123V)

FIG. 13—Phosphoprotein signals for CD2052, 24 hour incubation (ID-123V)

FIG. 14—Phosphoprotein signals for CD2059, 24 hour incubation (ID-123V)

FIG. 15—Phosphoprotein signals for CD2061, 24 hour incubation (ID-123V)

FIG. 16—Phosphoprotein signals for UC2075, 24 hour incubation (ID-123V)

FIG. 17—IL-6 production for IBD patient tissue samples (ID-123V)

FIG. 18—IL-8 production for IBD patient tissue samples (ID-123V)

FIG. 19—IL-10 production for IBD patient tissue samples (ID-123V)

FIG. 20—Phosphoprotein signals in Crohn's disease biopsy tissue (ID-142V, phosphoproteins ErbB1 to Ret)

FIG. 21—Phosphoprotein signals in Crohn's disease biopsy tissue (ID-142V, phosphoproteins ALK to AxI)

FIG. 22—Phosphoprotein signals in Crohn's disease biopsy tissue (ID-142V, phosphoproteins Tie2 to Stat3)

FIG. 23—Phosphoprotein signals (% inhibition vs control) in Crohn's disease biopsy tissue (ID-142V, phosphoproteins ErbB1 to Ret)

FIG. 24—Phosphoprotein signals (% inhibition vs control) in Crohn's disease biopsy tissue (ID-142V, phosphoproteins ALK to AxI)

FIG. 25—Phosphoprotein signals (% inhibition vs control) in Crohn's disease biopsy tissue (ID-142V, phosphoproteins Tie2 to Stat3)

FIG. 26—Phosphoprotein signals in ulcerative colitis biopsy tissue (ID-142V, phosphoproteins ErbB1 to Ret)

FIG. 27—Phosphoprotein signals in ulcerative colitis biopsy tissue (ID-142V, phosphoproteins ALK to AxI)

FIG. 28—Phosphoprotein signals in ulcerative colitis biopsy tissue (ID-142V, phosphoproteins Tie2 to Stat3)

FIG. 29—Cytokine production in Crohn's disease biopsy tissue (ID-142V, cytokines IFNg, IL-10 and TNF-alpha)

FIG. 30—Cytokine production in Crohn's disease biopsy tissue (ID-142V, cytokines IL-1b, and IL-17)

FIG. 31—Cytokine production in Crohn's disease biopsy tissue (ID-142V, cytokines IL-6 and IL-8)

FIG. 32—Cytokine production in Crohn's disease biopsy tissue relative to control (ID-142V, pro-inflammatory cytokines)

FIG. 33—Colon sample immunofluorescence displaying ID-123V

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1—Polypeptide sequence of ID-142V CDR1

SEQ ID NO: 2—Polypeptide sequence of ID-142V CDR2

SEQ ID NO: 3—Polypeptide sequence of ID-142V CDR3

SEQ ID NO: 4—Polypeptide sequence of ID-142V FR1

SEQ ID NO: 5—Polypeptide sequence of ID-142V FR2

SEQ ID NO: 6—Polypeptide sequence of ID-142V FR3

SEQ ID NO: 7—Polypeptide sequence of ID-142V FR4

SEQ ID NO: 8—Polypeptide sequence of ID-40V CDR1

SEQ ID NO: 9—Polypeptide sequence of ID-40V CDR2

SEQ ID NO: 10—Polypeptide sequence of ID-40V FR1

SEQ ID NO: 11—Polypeptide sequence of ID-40V FR2

SEQ ID NO: 12—Polypeptide sequence of ID-40V FR3

SEQ ID NO: 13—Polynucleotide sequence of 3′ primer

SEQ ID NO: 14—Polypeptide sequence of ID-40V CDR3

SEQ ID NO: 15—Polypeptide sequence of 5G9

SEQ ID NO: 16—Polypeptide sequence of ID-52V

SEQ ID NO: 17—Polypeptide sequence of ID-53V

SEQ ID NO: 18—Polypeptide sequence of ID-54V

SEQ ID NO: 19—Polypeptide sequence of ID-55V

SEQ ID NO: 20—Polypeptide sequence of ID-56V

SEQ ID NO: 21—Polypeptide sequence of ID-57V

SEQ ID NO: 22—Polypeptide sequence of ID-58V

SEQ ID NO: 23—Polypeptide sequence of ID-59V

SEQ ID NO: 24—Polypeptide sequence of ID-112V

SEQ ID NO: 25—Polypeptide sequence of ID-114V

SEQ ID NO: 26—Polypeptide sequence of ID-122V

SEQ ID NO: 27—Polypeptide sequence of ID-123V

SEQ ID NO: 28—Polypeptide sequence of ID-141V

SEQ ID NO: 29—Polypeptide sequence of ID-142V

SEQ ID NO: 30—Polypeptide sequence of ID-143V

SEQ ID NO: 31—Polypeptide sequence of ID-144V

SEQ ID NO: 32—Polypeptide sequence of 7F6

SEQ ID NO: 33—Polypeptide sequence of ID-3V

SEQ ID NO: 34—Polypeptide sequence of ID-6V

SEQ ID NO: 35—Polypeptide sequence of ID-40V

SEQ ID NO: 36—Polypeptide sequence of ID-47V

SEQ ID NO: 37—Polypeptide sequence of ID-49V

SEQ ID NO: 38—Polypeptide sequence of ID-50V

SEQ ID NO: 39—Polypeptide sequence of 21E6

SEQ ID NO: 40—Polypeptide sequence of 4D3

SEQ ID NO: 41—Polypeptide sequence of 3C12

SEQ ID NO: 42—Polypeptide sequence of 20A11

SEQ ID NO: 43—Polypeptide sequence of ID-74V

SEQ ID NO: 44—Polypeptide sequence of ID-75V

SEQ ID NO: 45—Polypeptide sequence of ID-2A

SEQ ID NO: 46—Polypeptide sequence of ID-58V CDR2

SEQ ID NO: 47—Polypeptide sequence of ID-59V CDR2

SEQ ID NO: 48—Polypeptide sequence of CDR3 of multiple ICVDs including 5G9

SEQ ID NO: 49—Polypeptide sequence of ID-53V CDR3

SEQ ID NO: 50—Polypeptide sequence of CDR3 of multiple ICVDs including ID-143V

SEQ ID NO: 51—Polypeptide sequence of 7F6 CDR3

SEQ ID NO: 52—Polypeptide sequence of ID-3V CDR3

SEQ ID NO: 53—Polypeptide sequence of ID-6V CDR3

SEQ ID NO: 54—Polynucleotide sequence encoding human IL-6R

SEQ ID NO: 55—Polypeptide sequence of ID-47V CDR3

SEQ ID NO: 56—Polypeptide sequence of ID-49V CDR3

SEQ ID NO: 57—Polypeptide sequence of ID-50V CDR3

SEQ ID NO: 58—Polypeptide sequence of 21E6 CDR3

SEQ ID NO: 59—Polynucleotide sequence encoding 5G9

SEQ ID NO: 60—Polynucleotide sequence encoding 5G9 (codon optimised for S. cerevisiae)

SEQ ID NO: 61—Polynucleotide sequence encoding 7F6

SEQ ID NO: 62—Polynucleotide sequence encoding 21E6

SEQ ID NO: 63—Polynucleotide sequence encoding ID-52V

SEQ ID NO: 64—Polynucleotide sequence encoding ID-53V

SEQ ID NO: 65—Polynucleotide sequence encoding ID-54V

SEQ ID NO: 66—Polynucleotide sequence encoding ID-55V

SEQ ID NO: 67—Polynucleotide sequence encoding ID-56V

SEQ ID NO: 68—Polynucleotide sequence encoding ID-57V

SEQ ID NO: 69—Polynucleotide sequence encoding ID-58V

SEQ ID NO: 70—Polynucleotide sequence encoding ID-59V

SEQ ID NO: 71—Polynucleotide sequence encoding ID-74V

SEQ ID NO: 72—Polynucleotide sequence encoding ID-75V

SEQ ID NO: 73—Polynucleotide sequence encoding ID-112V

SEQ ID NO: 74—Polynucleotide sequence encoding ID-114V

SEQ ID NO: 75—Polynucleotide sequence encoding ID-122V

SEQ ID NO: 76—Polynucleotide sequence encoding ID-123V

SEQ ID NO: 77—Polynucleotide sequence encoding ID-141V

SEQ ID NO: 78—Polynucleotide sequence encoding ID-142V

SEQ ID NO: 79—Polynucleotide sequence encoding ID-143V

SEQ ID NO: 80—Polynucleotide sequence encoding ID-144V

SEQ ID NO: 81—Polypeptide sequence of human IL-6R (NCBI Reference Sequence NP_000556.1)

SEQ ID NO: 82—Polypeptide sequence of mature human IL-6R (cleaved at L20)

SEQ ID NO: 83—Polypeptide sequence of soluble human IL-6R isoform produced by differential mRNA splicing

SEQ ID NO: 84—Polypeptide sequence of soluble human IL-6R isoform produced by protease shedding

SEQ ID NO: 85—Polypeptide sequence of predicted full length precursor cynomolgous monkey IL-6R (NCBI Reference Sequence: XP_005541720.1)

SEQ ID NO: 86—Polypeptide sequence of FR1 of ID-141V, ID-142V, ID-143V and ID-144V

SEQ ID NO: 87—Polypeptide sequence of FR2 of ID-141V, ID-142V, ID-143V and ID-144V

SEQ ID NO: 88—Polypeptide sequence of FR3 of ID-141V and ID-143V

SEQ ID NO: 89—Polypeptide sequence of FR3 of ID-142V and ID-144V

SEQ ID NO: 90—Polypeptide sequence of FR4 of ID-141V, ID-142V, ID-143V and ID-144V

SEQ ID NO: 91—Polypeptide VHHR sequence of 7F6

SEQ ID NO: 92—Polypeptide VHHR sequence of 5G9

SEQ ID NO: 93—Polypeptide VHHR sequence of 21E6

SEQ ID NO: 94—Polypeptide germline equivalent sequence of 7F6

SEQ ID NO: 95—Polypeptide germline equivalent sequence of 5G9

SEQ ID NO: 96—Polypeptide germline equivalent sequence of 21E6

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides including antibodies and antibody fragments including the VH and VHH A conventional antibody or immunoglobulin (Ig) is a protein comprising four polypeptide chains: two heavy (H) chains and two light (L) chains. Each chain is divided into a constant region and a variable domain. The heavy chain variable domains are abbreviated herein as VHC, and the light (L) chain variable domains are abbreviated herein as VLC. These domains, domains related thereto and domains derived therefrom, are referred to herein as immunoglobulin chain variable domains. The VHC and VLC domains can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDRs”), interspersed with regions that are more conserved, termed “framework regions” (“FRs”). The framework and complementarity determining regions have been precisely defined (Kabat et al., 1991, herein incorporated by reference in its entirety). In a conventional antibody, each VHC and VLC is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The conventional antibody tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains is formed with the heavy and the light immunoglobulin chains inter-connected by e.g. disulfide bonds, and the heavy chains similarity connected. The heavy chain constant region includes three domains, CH1, CH2 and CH3. The light chain constant region is comprised of one domain, CL. The variable domain of the heavy chains and the variable domain of the light chains are binding domains that interact with an antigen. The constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (C1q) of the classical complement system. The term antibody includes immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be kappa or lambda types. The overall structure of immunoglobulin-gamma (IgG) antibodies assembled from two identical heavy (H)-chain and two identical light (L)-chain polypeptides is well established and highly conserved in mammals (Padlan 1994).

An exception to conventional antibody structure is found in sera of Camelidae. In addition to conventional antibodies, these sera possess special IgG antibodies. These IgG antibodies, known as heavy-chain antibodies (HCAbs), are devoid of the L chain polypeptide and lack the first constant domain (CH1). At its N-terminal region, the H chain of the homodimeric protein contains a dedicated immunoglobulin chain variable domain, referred to as the VHH, which serves to associate with its cognate antigen (Muyldermans 2013. Hamers-Casterman et al., 1993, Muyldermans et al., 1994, herein incorporated by reference in their entirety).

An antigen-binding fragment (or “antibody fragment”, “immunoglobulin fragment” or “antigen-binding polypeptide”) as used herein refers to a portion of an antibody that specifically binds to IL-6R (e.g. a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to IL-6R). Examples of binding fragments encompassed within the term antigen-binding fragment include:

(i) a Fab fragment (a monovalent fragment consisting of the VLC, VHC, CL and CH1 domains); (ii) a F(ab′)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region); (iii) a Fd fragment (consisting of the VHC and CH1 domains); (iv) a Fv fragment (consisting of the VLC and VHC domains of a single arm of an antibody); (v) an scFv fragment (consisting of VLC and VHC domains joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VLC and VHC regions pair to form monovalent molecules); (vi) a VH (an immunoglobulin chain variable domain consisting of a VHC domain (Ward et al., 1989); (vii) a VL (an immunoglobulin chain variable domain consisting of a VLC domain); (viii) a V-NAR (an immunoglobulin chain variable domain consisting of a VHC domain from chondrichthyes IgNAR (Roux et al., 1998 and Griffiths et al., 2013, herein incorporated by reference in their entirety)

(ix) a VHH.

The total number of amino acid residues in a VHH or VH may be in the region of 110-140, is suitably 115-130, more suitably 120-125, most suitably 123.

Immunoglobulin chain variable domains of the invention may for example be obtained by preparing a nucleic acid encoding an immunoglobulin chain variable domain using techniques for nucleic acid synthesis, followed by expression of the nucleic acid thus obtained According to a specific embodiment, an immunoglobulin chain variable domain of the invention does not have an amino acid sequence which is exactly the same as (i.e. shares 100% sequence identity with) the amino acid sequence of a naturally occurring polypeptide such as a VH or VHH domain of a naturally occurring antibody.

The examples provided herein relate to immunoglobulin chain variable domains per se which bind to IL-6R. The principles of the invention disclosed herein are, however, equally applicable to any polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, such as antibodies and antibody fragments. For example, the anti-IL-6R immunoglobulin chain variable domains disclosed herein may be incorporated into a polypeptide such as a full length antibody. Such an approach is demonstrated by McCoy et al., 2014, who provide an anti-HIV VHH engineered as a fusion with a human Fc region (including hinge, CH2 and CH3 domains), expressed as a dimer construct.

Substituting at least one amino acid residue in the framework region of a non human immunoglobulin variable domain with the corresponding residue from a human variable domain is humanisation. Humanisation of a variable domain may reduce immunogenicity in humans.

Suitably, the polypeptide of the present invention consists of an immunoglobulin chain variable domain. Suitably, the polypeptide of the present invention is an antibody or an antibody fragment. Suitably the antibody fragment is a VHH, a VH, a VL, a V-NAR, a Fab fragment, a VL or a F(ab′)2 fragment (such as a VHH or VH, most suitably a VHH).

Specificity, Affinity, Avidity and Cross-Reactivity

Specificity refers to the number of different types of antigens or antigenic determinants to which a particular antigen-binding polypeptide can bind. The specificity of an antigen-binding polypeptide is the ability of the antigen-binding polypeptide to recognise a particular antigen as a unique molecular entity and distinguish it from another.

Affinity, represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding polypeptide (Kd), is a measure of the binding strength between an antigenic determinant and an antigen-binding site on the antigen-binding polypeptide: the lesser the value of the Kd, the stronger the binding strength between an antigenic determinant and the antigen-binding polypeptide (alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1/Kd). Affinity can be determined by known methods, depending on the specific antigen of interest.

Avidity is the measure of the strength of binding between an antigen-binding polypeptide and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antigen-binding polypeptide and the number of pertinent binding sites present on the antigen-binding polypeptide.

Suitably, antigen-binding polypeptides of the invention will bind with an equilibrium dissociation constant (Kd) of at least 1×10⁻⁶ M, more suitably at least 1×10⁻⁷ M, more suitably at least 1×10⁻⁸ M, more suitably at least 1×10⁻⁹ M.

Any Kd value less than 10⁻⁸ is considered to indicate binding. Specific binding of an antigen-binding polypeptide to an antigen or antigenic determinant can be determined in any suitable known manner, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known in the art.

An anti-IL-6R polypeptide, a polypeptide which interacts with IL-6R, or a polypeptide against IL-6R, are all effectively polypeptides which bind to IL-6R. A polypeptide of the invention may bind to a linear or conformational epitope on IL-6R. The term “binds to IL-6R” means binding to IL-6R wherein, for example, IL-6R may be comprised within the IL-6 receptor complex wherein the IL-6R receptor comprises gp130 and/or IL-6.

Suitably, the polypeptide of the invention will bind to both soluble and membrane IL-6R and more suitably with higher affinity to soluble IL-6R than membrane IL-6R. Suitably, the polypeptide of the invention will bind to human IL-6R. More suitably, the polypeptide of the invention will bind to both human and at least one additional primate IL-6R selected from the group consisting of baboon IL-6R, marmoset IL-6R, cynomolgus IL-6R and rhesus IL-6R. Most suitably, the polypeptide of the invention binds to both human and cynomolgus IL-6R.

Suitably, the polypeptide of the invention will neutralise both soluble and membrane IL-6R or more suitably only soluble IL-6R. Suitably, the polypeptide of the invention will neutralise human IL-6R. More suitably, the polypeptide of the invention will neutralise both human and at least one additional primate IL-6R selected from the group consisting of baboon IL-6R, marmoset IL-6R, cynomolgus IL-6R and rhesus IL-6R. Most suitably, the polypeptide of the invention neutralises both human and cynomolgus IL-6R.

Suitably, IL-6R is human or cynomolgous monkey soluble or membrane IL-6R. More suitably, IL-6R is IL-6R is human membrane or soluble IL-6R. More suitably, IL-6R is human soluble IL-6R.

Suitably IL-6R is a polypeptide comprising or more suitably consisting of any one of SEQ ID NO: 81-85. More suitably IL-6R is a polypeptide comprising or more suitably consisting of any one of SEQ ID NO: 81-84. Most suitably IL-6R is a polypeptide comprising or more suitably consisting of any one of SEQ ID NO: 82-84.

Polypeptides capable of reacting with IL-6R from humans and IL-6R from another species (“cross-reacting”), such as with cynomolgus monkey IL-6R, are advantageous because they allow preclinical studies to be more readily performed in animal models.

Suitably the polypeptide of the invention is directed against epitopes on IL-6R (and in particular epitopes on IL-6R which are exposed when IL-6R is comprised in an IL-6/IL-6R complex) that lie in and/or form part of the gp130-binding site(s) of the IL-6/IL-6R complex, such that said polypeptide of the invention, upon binding to IL-6R, results in inhibiting or reducing signalling mediated by the IL-6/IL-6R complex via gp130 association.

The polypeptides of the present invention bind to one or more epitope(s) on IL-6R. In one aspect of the invention there is provided a polypeptide which binds to at least a part of the same epitope on IL-6R, more suitably substantially the whole or the same epitope or most suitably the whole of the same epitope on IL-6R as 5G9, ID-52V, ID-53V, ID-54V, ID-55V, ID-56V, ID-57V, ID-58V, ID-59V, ID-112V, ID-114V, ID-122V, ID-123V, ID-141V, ID-142V, ID-143V, ID-144V, 7F6, ID-3V, ID-6V, ID-40V, ID-47V, ID-49V or ID-50V.

Suitably, the polypeptide of the invention is isolated. An “isolated” polypeptide is one that is removed from its original environment. For example, a naturally-occurring polypeptide of the invention is isolated if it is separated from some or all of the coexisting materials in the natural system.

Potency, Inhibition and Neutralisation

Potency is a measure of the activity of a therapeutic agent expressed in terms of the amount required to produce an effect of given intensity. A highly potent agent evokes a greater response at low concentrations compared to an agent of lower potency that evokes a smaller response at low concentrations. Potency is a function of affinity and efficacy. Efficacy refers to the ability of therapeutic agent to produce a biological response upon binding to a target ligand and the quantitative magnitude of this response. The term half maximal effective concentration (EC50) refers to the concentration of a therapeutic agent which causes a response halfway between the baseline and maximum after a specified exposure time. The therapeutic agent may cause inhibition or stimulation. It is commonly used, and is used herein, as a measure of potency.

A neutralising polypeptide for the purposes of the invention is a polypeptide which interferes in the binding of a sIL-6/IL-6R complex to gp130 as measured by ELISA. Alternatively, or in addition, a neutralising polypeptide for the purposes of the invention is a polypeptide which reduces the proliferation of cells presenting surface IL-6R (such as TF-1 cells) which are exposed to exogenous IL-6, by preventing formation of IL-6/IL-6R/gp130 cis-signalling complexes.

A method for determining the potency of an anti-IL-6R agent in neutralising IL-6R is as follows:

1. Standard gp130 ELISA Assay

The objective of this assay is to measure the potency of anti-IL-6R ICVDs by measuring interference in the binding to gp130 of a sIL-6/IL-6R complex. This assay detects binding of hIL-6R/hIL-6 complexes to recombinant human gp130. This interaction can be competitively inhibited by anti-IL-6R ICVDs, causing reduced binding of hIL-6R-hIL-6 complexes to gp130. Therefore, high signal in this ELISA represents a low concentration or low affinity of anti-IL-6R ICVD, and vice versa.

Materials Solutions Required: 1×PBS PBST (1×PBS, 0.05% Tween 20)

Block buffer (1% BSA in 1×PBS, pH 7.3-7.5) 0.5 M Sulphuric acid (H₂SO₄)

Reagents Required:

Recombinant soluble human gp130 at known concentration ICVD stock (preferably 250 ug/mL diluted to 20 ug/mL in protease testing solution) Recombinant soluble human IL-6 at known concentration Recombinant soluble human IL-6R at known concentration Biotinylated goat anti-IL-6R polyclonal antibody (R&D systems BAF227); resuspended at 250 ug/ml in sterile PBS.

ExtrAvidin-Peroxidase (Sigma E2886)

TMB substrate (Microwell Peroxidase substrate System 2-C, KPL, 50-70-00)

Procedure Preparation:

-   -   1. Determine number of plates required for the assay.     -   2. Prepare the relevant volume (up to 3 plates at a time) of 0.2         μg/ml recombinant soluble human gp130 in PBS with 5 ug/mL BSA in         1×PBS.     -   3. Working quickly, dispense 50 μl/well into Maxisorp 96-well         ELISA plates (Nunc), loading a maximum of 3 plates in one batch.     -   4. Shake plate briefly, seal and incubate at 4° C. overnight.

Assay:

1. Wash the ELISA plate using a plate washer (4×˜380 μl PBST). Bang the plate on towel to remove residual liquid. 2. Apply 200 μl/well block buffer. Seal and incubate on a rotary plate shaker for ≥1 hour. 3. Prepare a dilution series of ICVD standards between 0.004 nM to 80 nM in minimum final volumes of 70 μl using block buffer as a diluent. 4. Prepare appropriate dilutions of samples to be tested in block buffer, such that their estimated final concentration on the plate will fall in the range of 0.001 nM to 250 nM ICVD. 5. Prepare a 40 ng/ml IL-6R solution in block buffer. 6. In a separate 96-well plate, mix together 50 μl of each ICVD dilution with 50 μl IL-6R solution. In each dilution series include one well with no ICVD. Incubate for 1 hour on a rotary plate shaker. 7. Prepare a 100 ng/ml IL-6 solution in block buffer. 8. In a further additional 96-well plate, mix together 85 μl ICVD-IL-6R mixture from step 6 with 85 μl IL-6 solution prepared in step 7. Include wells containing block buffer only, such that the following controls are applied to each plate: IL-6 only, and no ICVD (IL6+IL6R only). Incubate for 10 minutes on rotary plate shaker. 9. Wash blocked ELISA plate as in step 1. 10. Transfer 50 μl of the mixtures prepared in step 8 to the washed ELISA plate in triplicate. Seal and incubate on a rotary plate shaker for 2 hours. 11. Wash blocked ELISA plate as in step 1. 12. Prepare 5.2 ml/plate 125 ug/mL of BAF227 anti-hIL-6R antibody made up in block buffer. Add 50 μl/well, seal, shake briefly, and incubate for 1 hour at room temperature or overnight at 4° C. 13. Wash blocked ELISA plate as in step 1. 14. Prepare 5.2 ml/plate of 1/1,000-1/3000 dilution of Extravidin in block buffer. Add 50 μl/well, seal, and incubate on a rotary shaker for 30 mins. 15. Wash blocked ELISA plate as in step 1. 16. Prepare 10 ml/plate TMB substrate (1:1 ratio of substrate A and B). Add 100 μl/well, seal and incubate on a rotary plate shaker until a mid blue colour evolves in the lowest dilution wells or up to a maximum of 30 mins. Shield from light. 17. Stop reaction with 50 μl/well 0.5 M H₂SO₄. 18. Read plate at 450 nm. 19. Use standard curve to interpolate concentrations of active ICVD. Raw OD450 values are adjusted with readings taken from blank control wells. Standard curves are plotted using appropriate software (e.g. Graphpad Prism using Log(inhibitor) vs. response—variable slope (four parameters)). ICVD concentrations in the test samples are calculated in the software using the standard curve.

A method for determining the potency of an anti-IL-6R agent in neutralising cynomolgous monkey IL-6R is as follows:

2. Standard Anti-Cynomolgus Monkey IL-6R gp30 ELISA Assay

The objective of this assay is to measure the potency of anti-IL-6R antibodies in neutralising cynomolgus monkey IL-6R activity by inhibiting the formation of a cynomolgus monkey IL-6R, human gp130, and human IL-6 complex. This assay detects binding of IL-6R/hIL-6 complexes to recombinant human gp130. This interaction can be competitively inhibited by anti-IL-6R ICVDs, causing reduced binding of IL-6R/hIL-6 complexes to gp130. Therefore, high signal in this ELISA represents a low concentration or low affinity of anti-IL-6R ICVD, and vice versa.

Materials Solutions Required: 1×PBS 1% BSA in PBS PBST (1×PBS, 0.05% Tween 20)

Block buffer (1% BSA in 1×PBS, pH 7.3-7.5) Assay buffer (1% BSA, 2× protease inhibitor* in 1×PBS) 0.5 M Sulphuric acid (H₂SO₄) *2× protease inhibitor=1 tablet per 50 ml buffer

Reagents Required:

Recombinant soluble human gp130 at known concentration SigmaFast protease inhibitor tablets (S8820) ICVD stock of known concentration Recombinant soluble human IL-6 at known concentration Recombinant soluble human IL-6R at known concentration Cynomolgus monkey serum Biotinylated goat anti-IL-6R polyclonal antibody (R&D systems BAF227); resuspended at 250 ug/ml in sterile PBS.

ExtrAvidin-Peroxidase (Sigma E2886)

TMB substrate (Microwell Peroxidase substrate System 2-C, KPL, 50-70-00)

Procedure Preparation:

-   -   1. Determine number of plates required for the assay.     -   2. Prepare the relevant volume (up to 3 plates at a time) of 1         pg/ml recombinant soluble human gp130 in PBS+5 μg/ml BSA, which         are for use for the cynomolgus monkey serum. Coat the same         number of plates with 0.2 ug/mL gp130 with +5 μg/ml BSA for the         human IL6R control.     -   3. Working quickly, dispense 50 μl/well into Maxisorp 96-well         ELISA plates (Nunc), loading a maximum of 4 plates in one batch.     -   4. Shake plate briefly, seal and incubate at 4° C. overnight.

Assay:

1. Wash the ELISA plate using a plate washer (4×˜380 μl PBST). Bang the plate on towel to remove residual liquid. 2. Apply 200 μl/well block buffer. Seal and incubate on a rotary plate shaker for ≥1 hour. 3. Prepare a dilution series of ICVD standards between 0.004 nM to 1000 nM in minimum final volumes of 70 μl using assay buffer as a diluent. 4. Prepare a 20 ng/ml IL-6R solution in assay buffer. 5. Prepare a 2.5× dilution of normal cynomolgus monkey serum in assay buffer. 6. In a separate 96-well plate, mix together 50 μl of each ICVD dilution with 50 μl IL-6R solution or monkey serum. In each dilution series include one well with no ICVD. Incubate for 1 hour on a rotary plate shaker. 7. Prepare a 100 ng/ml IL-6 solution in block buffer. 8. In a further additional 96-well plate, mix together 85 μl ICVD-IL-6R mixture from step 6 with 85 μl IL-6 solution prepared in step 7. Include wells containing block buffer only, such that the following controls are applied to each plate: IL-6 only, and no ICVD (IL6+IL6R only). Incubate for 10 minutes on rotary plate shaker. 9. Wash blocked ELISA plate as in step 1. 10. Transfer 50 μl of the mixtures prepared in step 8 to the washed ELISA plate in triplicate. Seal and incubate on a rotary plate shaker for 2 hours. 11. Wash blocked ELISA plate as in step 1. 12. Prepare 5.2 ml/plate 125 ug/mL of BAF227 anti-hIL-6R antibody made up in block buffer. Add 50 μl/well, seal, shake briefly, and incubate overnight at 4° C. 13. Wash blocked ELISA plate as in step 1. 14. Prepare 5.2 ml/plate of 1/3000 dilution of Extravidin in block buffer. Add 50 μl/well, seal, and incubate on a rotary shaker for 30 mins. 15. Wash blocked ELISA plate as in step 1. 16. Prepare 10 ml/plate TMB substrate (1:1 ratio of substrate A and B). Add 100 μl/well, seal and incubate on a rotary plate shaker until a mid blue colour evolves in the lowest dilution wells or up to a maximum of 30 mins. Shield from light. 17. Stop reaction with 50 μl/well 0.5 M H₂SO₄. 18. Read plate at 450 nm. 19. Use standard curve to interpolate concentrations of active ICVD. Raw OD450 values are adjusted with readings taken from blank control wells. Standard curves are plotted using appropriate software (e.g. Graphpad Prism using Log(inhibitor) vs. response—variable slope (four parameters)). ICVD concentrations in the test samples are calculated in the software using the standard curve.

A method for determining the potency of an anti-IL-6R agent in inhibiting membrane bound IL-6R mediated IL-6 cis-signalling is as follows:

3. The Standard TF1 Cell Assay

This assay detects the activity of exogenous IL-6 on TF1 cell proliferation. TF1 cells are a human erythroblast immortal cell line that expresses the membrane-bound IL-6 receptor on their surface. In the presence of exogenously added IL-6, the cells are stimulated via the IL-6 cis-signalling pathway to proliferate. Anti membrane-bound IL-6R compounds prevent the formation of the complete IL-6/IL-6R/gp130 complex that is required for signalling, reducing IL-6-stimulated proliferation in dose-dependent manner. Cell proliferation is measured after two days using resazurin, a blue dye that is irreversibly reduced to the pink coloured and highly red fluorescent resorufin by cellular metabolic processes.

Materials Standard Components:

Cell line: TF-1 (Public Health England, Cat. No. 93022307). Exponentially growing cells (<5×10̂5 cells/mL) cultivated in Advanced RPMI with 5% heat-inactivated foetal bovine serum and 2.5 ng/mL GM-CSF (Human Granulocyte Macrophage Colony Stimulating Factor). Assay medium: Advanced RMPI (LifeTech 12633-012) with 2 mM L-glutamine, Pen/Strep 1×PBS

3% SDS

Sterile MQ water Sterile 96 well micro-titre plates for cell culture, flat bottomed, optically clear 12 multi-channel pipette and filtered tips

Reagents:

Recombinant soluble human IL-6 at known concentration Anti-IL-6R antibodies or ICVD of known concentration

Resazurin (Alamar Blue; LifeTech, DAL1100) Procedure Cell Preparation:

-   -   a. Pre-warm media and 1×PBS in a 37° C. water bath.     -   b. Determine the number of plates required for the assay.     -   c. Fill the outside wells with 320 uL of sterile MilliQ water.     -   d. Fill the spaces between the wells with 160 uL of the same         water.     -   e. Calculate the volume of the cell culture you will need to         have 20,000 cells/well using the equation:

(60 wells per plate×#plates×20,000 cells/well×1.2(for 20% overage)=total cells Cell count(cells/ml)

-   -   f. Transfer cells to falcon tube. Take to maximum tube volume         with 1×PBS. Spin 5′ at 1.1 k×rpm at 20 degrees C.     -   g. Remove medium from cell pellet with a 10 mL stripette, leave         ˜200 uL on the cells, ensuring no cells are aspirated. Tap cells         in residual volume to resuspend.     -   h. Wash cells in full tube volume of 1×PBS spin as before.     -   i. Remove 1×PBS with a 10 mL stripette, leave ˜200 uL on the         cells, ensuring no cells are aspirated. Remove residual with         P200.     -   j. Resuspend cell pellet by tapping then add assay medium using         the equation below to calculate the volume required:

60 wells per plate×#plates×0.05 mL volume per well×1.2(for 20% overage)=total volume

-   -   k. Fill plates with 50 ul cells per well proceeding row by row.         Place filled plates in the incubator. Agitate the cells by         pipetting or shaking the trough intermittently during this         process.

ICVD/Antibody Preparation:

-   -   a. Prepare the assay stock of IL-6 at 20 ng/mL (4× the assay         concentration) in a volume of assay medium sufficient to add 25         ul per well.     -   b. Prepare a 9 point serial dilution series of ICVD reference         standards at 4× the assay concentration (being between 0.04 nM         and 2500 nM in minimum final volumes of 100 μl using assay         medium as a diluent. In a tenth well, add assay medium only. For         example, starting in well A1 make the dilutions series along to         plate to well A9. In well A10, add medium only.     -   c. In a fresh microtitre plate, add a minimum volume of 85 uL of         IL-6 to the equivalent 9 wells used in the dilution series. In         the tenth well add either assay medium or IL-6 to the effect         that each final assay plate will have wells with IL-6 only         (maximum proliferation) and media only (minimum proliferation).     -   d. To the IL-6 mix plate prepared above, add an equivalent         volume (e.g. 85 uL) of each ICVD/antibody serial dilution. At         this stage both IL-6 and ICVD will be at 2× the assay         concentration.

Assay:

-   -   a. Mix the ICVD/Antibody & IL-6 mixtures by pipetting and add 50         ul of each mix to the cells.     -   b. Incubate the cells for 2 days at 37° C., 5% CO₂.     -   c. Add 10 uL of AlamarBlue to cells. Protect from light.     -   d. Shake the cells gently for 30 s on a plate shaker to mix     -   e. Incubate 37° C., 5% CO₂ for 2 hours.     -   f. Stop cellular processes with 50 uL 3% SDS. Protect from light     -   g. Read on plate reader (e.g. BMG Fluorstar) at Ex 544 nm-Em 590         nm     -   h. Use calibration curve to interpolate unknown sample         concentrations. Raw OD450 values are adjusted with readings         taken from blank control wells. Standard curves are plotted         using appropriate software (e.g. Graphpad Prism using         Log(inhibitor) vs. response-variable slope (four parameters)).         ICVD concentrations in the test samples are calculated in the         software using the standard curve.

Suitably the polypeptide or construct of the invention neutralizes sIL-6R-IL-6 binding to gp130 in an ELISA assay such as the standard gp130 ELISA with an EC50 of 100 nM or less, such as 75 nM or less, such as 50 nM or less, such as 40 nM or less, such as 30 nM or less, such as 25 nM or less, such as 20 nM or less, such as 10 nM or less, such as 5 nM or less, such as 2 nM or less, such as 1.5 nM or less, such as 1 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.7 nM or less, such as 0.6 nM or less, such as 0.5 nM or less, such as 0.45 nM or less, such as 0.4 nM or less, such as 0.35 nM or less, such as 0.3 nM or less, such as 0.25 nM or less, such as 0.2 nM or less, such as 0.16 nM or less, such as 0.15 nM or less, such as 0.1 nM or less.

Suitably the polypeptide or construct of the invention neutralizes cynomolgous IL-6R-human IL-6 binding to human gp130 in an ELISA assay such as the standard anti-cynomolgus monkey IL-6R gp130 ELISA assay with an EC50 of 20 nM or less, such as 10 nM or less, such as 5 nM or less, such as 2 nM or less, such as 1.5 nM or less, such as 1 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.7 nM or less, such as 0.6 nM or less, such as 0.5 nM or less, such as 0.45 nM or less, such as 0.4 nM or less, such as 0.35 nM or less, such as 0.3 nM or less, such as 0.25 nM or less, such as 0.2 nM or less, such as 0.16 nM or less, such as 0.15 nM or less, such as 0.1 nM or less.

Suitably the polypeptide or construct of the invention prevents proliferation of TF-1 cells in the standard TF-1 assay with an EC₅₀ of 20 nM or less, such as 10 nM or less, such as 5 nM or less, such as 2 nM or less, such as 1.5 nM or less, such as 1 nM or less, such as 0.9 nM or less, such as 0.8 nM or less, such as 0.7 nM or less, such as 0.6 nM or less, such as 0.5 nM or less, such as 0.45 nM or less, such as 0.4 nM or less, such as 0.35 nM or less, such as 0.3 nM or less, such as 0.25 nM or less, such as 0.2 nM or less, such as 0.16 nM or less, such as 0.15 nM or less, such as 0.1 nM or less.

Suitably the polypeptide of the invention inhibits IL-6R trans-signalling to a higher extent than it inhibits IL-6R cis-signalling, i.e. the polypeptide is ‘trans-selective’.

Polypeptide and Polynucleotide Sequences

For the purposes of comparing two closely-related polypeptide sequences, the “% sequence identity” between a first polypeptide sequence and a second polypeptide sequence may be calculated using NCBI BLAST v2.0, using standard settings for polypeptide sequences (BLASTP). For the purposes of comparing two closely-related polynucleotide sequences, the “% sequence identity” between a first nucleotide sequence and a second nucleotide sequence may be calculated using NCBI BLAST v2.0, using standard settings for nucleotide sequences (BLASTN).

Polypeptide or polynucleotide sequences are said to be the same as or identical to other polypeptide or polynucleotide sequences, if they share 100% sequence identity over their entire length. Residues in sequences are numbered from left to right, i.e. from N- to C-terminus for polypeptides; from 5′ to 3′ terminus for polynucleotides.

A “difference” between sequences refers to an insertion, deletion or substitution of a single amino acid residue in a position of the second sequence, compared to the first sequence. Two polypeptide sequences can contain one, two or more such amino acid differences. Insertions, deletions or substitutions in a second sequence which is otherwise identical (100% sequence identity) to a first sequence result in reduced % sequence identity. For example, if the identical sequences are 9 amino acid residues long, one substitution in the second sequence results in a sequence identity of 88.9%. If the identical sequences are 17 amino acid residues long, two substitutions in the second sequence results in a sequence identity of 88.2%. If the identical sequences are 7 amino acid residues long, three substitutions in the second sequence results in a sequence identity of 57.1%. If first and second polypeptide sequences are 9 amino acid residues long and share 6 identical residues, the first and second polypeptide sequences share greater than 66% identity (the first and second polypeptide sequences share 66.7% identity). If first and second polypeptide sequences are 17 amino acid residues long and share 16 identical residues, the first and second polypeptide sequences share greater than 94% identity (the first and second polypeptide sequences share 94.1% identity). If first and second polypeptide sequences are 7 amino acid residues long and share 3 identical residues, the first and second polypeptide sequences share greater than 42% identity (the first and second polypeptide sequences share 42.9% identity).

Alternatively, for the purposes of comparing a first, reference polypeptide sequence to a second, comparison polypeptide sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one amino acid residue into the sequence of the first polypeptide (including addition at either terminus of the first polypeptide). A substitution is the substitution of one amino acid residue in the sequence of the first polypeptide with one different amino acid residue. A deletion is the deletion of one amino acid residue from the sequence of the first polypeptide (including deletion at either terminus of the first polypeptide).

For the purposes of comparing a first, reference polynucleotide sequence to a second, comparison polynucleotide sequence, the number of additions, substitutions and/or deletions made to the first sequence to produce the second sequence may be ascertained. An addition is the addition of one nucleotide residue into the sequence of the first polynucleotide (including addition at either terminus of the first polynucleotide). A substitution is the substitution of one nucleotide residue in the sequence of the first polynucleotide with one different nucleotide residue. A deletion is the deletion of one nucleotide residue from the sequence of the first polynucleotide (including deletion at either terminus of the first polynucleotide).

A “conservative” amino acid substitution is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue of similar chemical structure and which is expected to have little influence on the function, activity or other biological properties of the polypeptide. Such conservative substitutions suitably are substitutions in which one amino acid within the following groups is substituted by another amino acid residue from within the same group:

Group Amino acid residue Non-polar aliphatic Glycine Alanine Valine Leucine Isoleucine Aromatic Phenylalanine Tyrosine Tryptophan Polar uncharged Serine Threonine Asparagine Glutamine Negatively charged Aspartate Glutamate Positively charged Lysine Arginine

Suitably, a hydrophobic amino acid residue is a non-polar amino acid. More suitably, a hydrophobic amino acid residue is selected from V, I, L, M, F, W or C.

As used herein, numbering of polypeptide sequences and definitions of CDRs and FRs are as defined according to the Kabat system (Kabat et al., 1991, herein incorporated by reference in its entirety). A “corresponding” amino acid residue between a first and second polypeptide sequence is an amino acid residue in a first sequence which shares the same position according to the Kabat system with an amino acid residue in a second sequence, whilst the amino acid residue in the second sequence may differ in identity from the first. Suitably corresponding residues will share the same number (and letter) if the framework and CDRs are the same length according to Kabat definition. Alignment can be achieved manually or by using, for example, a known computer algorithm for sequence alignment such as NCBI BLAST v2.0 (BLASTP or BLASTN) using standard settings.

Polypeptide Sequences

Suitably, the polypeptide sequence of the present invention contains at least one alteration with respect to a native sequence. Suitably, the polynucleotide sequences of the present invention contain at least one alteration with respect to a native sequence. Suitably the alteration to the polypeptide sequence or polynucleotide sequence is made to increase stability of the polypeptide or encoded polypeptide to proteases present in the intestinal tract (for example trypsin and chymotrypsin).

The Kabat Numbering System Applied to Particular Immunoglobulin Chain Variable Domains of the Invention:

Note that the ICVDs of the invention provided above all share similar CDR3s. It should also be noted that the 5G9-related ICVDs above all share a ‘NIN’ insert in FR1 (29A-29C in Kabat numbering).

The Kabat Numbering System Applied to Other Immunoglobulin Chain Variable Domains Referred to in the Examples:

Note that the ICVDs above which fall outside the scope of the invention share distinct CDR3s from ICVDs falling within the scope of the invention. The CDRs of the polypeptides labelled above as ‘CDR-H1’/‘CDR1-H1’, ‘CDR-H2’/‘CDR2-H2’ and ‘CDR-H3’/‘CDR3-H3’ relate to ‘CDR1’, ‘CDR2’ and ‘CDR3’ respectively, as discussed herein. Numbering in the figures above with the prefix ‘H’ is Kabat numbering, while numbering below the amino acid sequence is numbering of amino acids consecutively from N- to C-terminus. The residues of each CDR or FR can also be numbered from the N- to the C-terminus of that CDR or FR. For example, it can be seen that CDR3 of 5G9 contains S at position 107 numbered from N- to C-terminus of the full length polypeptide, which is also position 100A according to Kabat, which is also residue number 7 of CDR3 of 5G9.

Also highlighted above are insertion sequences NIN (H29A-H29C), NSL (H82A-H82C), SPFN/SPHN (H100A-H100D and H100A-H100B (LA).

CDR3 Sequences

Suitably CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, more suitably 65%, 70%, 75%, 80%, 85%, 90% or greater sequence identity with SEQ ID NO: 3.

Alternatively, CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 3. Suitably, CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 3. Suitably, CDR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 3. Suitably, any substitutions are conservative, with respect to their corresponding residues in SEQ ID NO: 3.

Suitably any residues of CDR3 differing from their corresponding residues in SEQ ID NO: 3 are conservative substitutions with respect to their corresponding residues.

Suitably the residue of CDR3 corresponding to residue number 5 of SEQ ID NO: 3 is R or H, most suitably H. Suitably the residue of CDR3 corresponding to residue number 5 of SEQ ID NO: 3 is R or H, most suitably H, and any other residues of CDR3 differing from their corresponding residues in SEQ ID NO: 3 are conservative substitutions with respect to their corresponding residues.

Suitably the residue of CDR3 corresponding to residue number 9 of SEQ ID NO: 3 is F, L or H, more suitably F or H, most suitably H. Suitably the residue of CDR3 corresponding to residue number 9 of SEQ ID NO: 3 is F, L or H, most suitably H, and any other residues of CDR3 differing from their corresponding residues in SEQ ID NO: 3 are conservative substitutions with respect to their corresponding residues.

Suitably the residue of CDR3 corresponding to residue number 11 of SEQ ID NO: 3 is A, G, or a conservative substitution thereof. Suitably the residue of CDR3 corresponding to residue number 11 of SEQ ID NO: 3 is A or G, most suitably A. Suitably the residue of CDR3 corresponding to residue number 11 of SEQ ID NO: 3 is A or G, most suitably A, and any other residues of CDR3 differing from their corresponding residues in SEQ ID NO: 3 are conservative substitutions with respect to their corresponding residues.

Suitably CDR3 comprises or more suitably consists of SEQ ID NO: 3.

CDR1 and CDR2 Sequences Derived from 5G9

Suitably CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater sequence identity, with SEQ ID NO: 1.

Alternatively, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 1. Suitably, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 1. Suitably, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 1.

Suitably any residues of CDR1 differing from their corresponding residues in SEQ ID NO: 1 are conservative substitutions with respect to their corresponding residues. Suitably CDR1 comprises or more suitably consists of SEQ ID NO: 1.

Suitably CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater sequence identity, with SEQ ID NO: 2.

Alternatively, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 2. Suitably, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 2. Suitably, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 2.

Suitably any residues of CDR2 differing from their corresponding residues in SEQ ID NO: 2 are conservative substitutions with respect to their corresponding residues. Suitably CDR2 comprises or more suitably consists of SEQ ID NO: 2.

CDR1 and CDR2 Sequences Derived from 7F6

Suitably CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 20%, 40%, 60%, 80% or greater sequence identity, with SEQ ID NO: 8.

Alternatively, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 8. Suitably, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 8. Suitably, CDR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 8.

Suitably any residues of CDR1 differing from their corresponding residues in SEQ ID NO: 8 are conservative substitutions with respect to their corresponding residues. Suitably CDR1 comprises or more suitably consists of SEQ ID NO: 8.

Suitably CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater sequence identity, with SEQ ID NO: 9.

Alternatively, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 9. Suitably, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 9. Suitably, CDR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 9.

Suitably any residues of CDR2 differing from their corresponding residues in SEQ ID NO: 9 are conservative substitutions with respect to their corresponding residues. Suitably CDR2 comprises or more suitably consists of SEQ ID NO: 9.

Specific CDR Sequences

Some particularly suitable CDR sequences are shown in the table below. Suitably, CDR1 of the polypeptide of the invention is one of the CDR1 sequences listed below. Suitably, CDR2 of the polypeptide of the invention is one of the CDR2 sequences listed below. Suitably, CDR3 of the polypeptide of the invention is one of the CDR3 sequences listed below. Suitably, the polypeptide of the invention comprises a combination of two, or more suitably three, of the CDR sequences listed below.

Particular CDRs of the polypeptide of the invention:

CDR1 CDR2 CDR3 INVMA IIGKGGGTNYADFVKG DYEDRDSPFNAS (CDR1 of   (CDR2 of   (CDR3 of   multiple ICVDs multiple ICVDs multiple ICVDs including  including  including 5G9, ID-142V, ID-142V, SEQ ID NO: 48) SEQ ID NO: 1) SEQ ID NO: 2) INVIG IIGKGGGTNYADVVKG DYEDHDSPHNAS (CDR1 of  (ID-58V CDR2, (CDR3 of  multiple ICVDs SEQ ID NO: 46) multiple ICVDs including  including  ID-40V, ID-142V, SEQ ID NO: 8) SEQ ID NO: 3) — IIGKGGGTNDADFVKG DYEDRDSPHNAS (ID-59V CDR2, (ID-53V CDR3, SEQ ID NO: 47) SEQ ID NO: 49) — MIGRGEGANYGDFAKG DYEDHDSPFNAS (CDR2 of  (CDR3 of  multiple ICVDs multiple ICVDs including  including  ID-40V, ID-143V, SEQ ID NO: 9) SEQ ID NO: 50) — — DYEDRDSPFNGS (7F6 CDR3, SEQ ID NO: 51) — — DYEDHDSPFNGS (ID-3V CDR3, SEQ ID NO: 52) — — DYEDRDSPHNGS (ID-6V CDR3, SEQ ID NO: 53) — — DYEDHDSPHNGS (ID-40V CDR3, SEQ ID NO: 14) — — DYEDRDSPINGS (ID-47V CDR3, SEQ ID NO: 55) — — DYEDRDSPTNGS (ID-49V CDR3, SEQ ID NO: 56) — — DYEDRDSPVNGS (ID-50V CDR3, SEQ ID NO: 57) — — DYEDRDSPLNGS (21E6 CDR3, SEQ ID NO: 58)

Framework Sequences

Suitably FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 5%, 12%, 18%, 26%, 32%, 38%, 46%, 52%, 58%, 62%, 66%, 68%, 72%, 75%, 78%, 82%, 85%, 90%, 95% or greater sequence identity, with SEQ ID NO: 4 or SEQ ID NO: 10.

Alternatively, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 4 or SEQ ID NO: 10. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 4 or SEQ ID NO: 10. Suitably, FR1 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 28, more suitably no more than 26, more suitably no more than 24, more suitably no more than 22, more suitably no more than 20, more suitably no more than 18, more suitably no more than 16, more suitably no more than 14, more suitably no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 4 or SEQ ID NO: 10.

Suitably any residues of FR1 differing from their corresponding residues in SEQ ID NO: 4 or SEQ ID NO: 10 are conservative substitutions with respect to their corresponding residues.

Suitably the residue of FR1 corresponding to residue number 1 of SEQ ID NO: 4 or SEQ ID NO: 10 is G, A, V, L, I, F, P, S, T, Y, C, M, K, R, H, W, D, E or N (more suitably D or E, most suitably D). Suitably the residue of FR1 corresponding to residue number 23 of SEQ ID NO: 4 or SEQ ID NO: 10 is K or L. Suitably the residues of FR1 corresponding to residue numbers 2 to 5 of SEQ ID NO: 4 or SEQ ID NO: 10 are VQLV. Suitably the residue of FR1 corresponding to residue number 29 of SEQ ID NO: 4 or SEQ ID NO: 10 is F or S, most suitably S. Suitably the residue of FR1 corresponding to residue number 30 of SEQ ID NO: 4 or SEQ ID NO: 10 is N or S. Suitably FR1 comprises or more suitably consists of SEQ ID NO: 4 or SEQ ID NO: 10.

Suitably FR1 comprises the sequence NIN or three consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NIN. Most suitably FR1 comprises the sequence NIN. More suitably the last four C-terminal residues of FR1 are NINX or four consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NINX, wherein X is any amino acid. Suitably X is S or a conservative substitution of S, most suitably X is S. More suitably FR1 consists of 33 residues and the last four residues of FR1 are NINS.

Suitably FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 10%, 15%, 25%, 30%, 40%, 45%, 55%, 60%, 70%, 75%, 85%, 90% or greater sequence identity, with SEQ ID NO: 5 or SEQ ID NO: 11.

Alternatively, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 5 or SEQ ID NO: 11. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 5 or SEQ ID NO: 11. Suitably, FR2 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 13, more suitably no more than 12, more suitably no more than 11, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 5 or SEQ ID NO: 11.

Suitably any residues of FR2 differing from their corresponding residues in SEQ ID NO: 5 or SEQ ID NO: 11 are conservative substitutions with respect to their corresponding residues. Suitably the residue of FR2 corresponding to residue number 9 of SEQ ID NO: 5 or SEQ ID NO: 11 is G or Q. Suitably the residues of FR2 corresponding to residue numbers 8 to 11 of SEQ ID NO: 5 or SEQ ID NO: 11 are KERE, KELE, KGRE or KQRE; most suitably KGRE or KQRE. Suitably FR2 comprises or more suitably consists of SEQ ID NO: 5 or SEQ ID NO: 11.

Suitably FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 8%, 15%, 20%, 26%, 32%, 40%, 45%, 52%, 58%, 65%, 70%, 76%, 80%, 82%, 85%, 90%, 92%, 95% or greater sequence identity, with SEQ ID NO: 6 or SEQ ID NO: 12.

Alternatively, FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 6 or SEQ ID NO: 12. Suitably, FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 6 or SEQ ID NO: 12. Suitably, FR3 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 29, more suitably no more than 27, more suitably no more than 25, more suitably no more than 23, more suitably no more than 21, more suitably no more than 19, more suitably no more than 17, more suitably no more than 15, more suitably no more than 13, more suitably no more than 11, more suitably no more than 9, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 6 or SEQ ID NO: 12.

Suitably the residue of FR3 corresponding to residue number 8 of SEQ ID NO: 6 or SEQ ID NO: 12 is an amino acid which is hydrophobic (suitably A or N, more suitably A). Suitably the residue of FR3 corresponding to residue number 9 of SEQ ID NO: 6 or SEQ ID NO: 12 is an amino acid which is hydrophobic (suitably A or S, more suitably A). Suitably the residue of FR3 corresponding to residue number 13 of SEQ ID NO: 6 or SEQ ID NO: 12 is V or L, most suitably V. Suitably the residue of FR3 corresponding to residue number 14 of SEQ ID NO: 6 or SEQ ID NO: 12 is N or Y, most suitably Y. Suitably the residue of FR3 corresponding to residue number 21 of SEQ ID NO: 6 or SEQ ID NO: 12 is R or K, most suitably R.

Suitably any residues of FR3 differing from their corresponding residues in SEQ ID NO: 6 or SEQ ID NO: 12 are conservative substitutions with respect to their corresponding residues. Suitably FR3 comprises or more suitably consists of SEQ ID NO: 6 or SEQ ID NO: 12.

Suitably FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater sequence identity, with SEQ ID NO: 7.

Alternatively, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NO: 7. Suitably, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NO: 7. Suitably, FR4 of the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to SEQ ID NO: 7.

Suitably any residues of FR4 differing from their corresponding residues in SEQ ID NO: 7 are conservative substitutions with respect to their corresponding residues. Suitably FR4 comprises or more suitably consists of SEQ ID NO: 7.

The particularly preferred framework sequences of ICVDs ID-141V, ID-142V, ID-143V and ID-144V are listed below.

(FR1 of ID-141V, ID-142V, ID-143V and ID-144V, SEQ ID NO: 86) DVQLVESGGGLVQAGGSTRLTCKASGSISNINS (FR2 of ID-141V, ID-142V, ID-143V and ID-144V, SEQ ID NO: 87) WYRQAPGKGRELVA (FR3 of ID-141V and ID-143V, SEQ ID NO: 88) RFTISRDAAKNTLYLQMNSLRPEDTAVYYCYA (FR3 of ID-142V and ID-144V, SEQ ID NO: 89) RFTISRDAAKNTVYLQMNSLRPEDTAVYYCYA (FR4 of ID-141V, ID-142V, ID-143V and ID-144V, SEQ ID NO: 90) WGQGTQVTVSS

Suitably FR1 of the polypeptide of the invention comprises or more suitably consists of SEQ ID NO: 86. Suitably FR2 of the polypeptide of the invention comprises or more suitably consists of SEQ ID NO: 87. Suitably FR3 of the polypeptide of the invention comprises or more suitably consists of SEQ ID NO: 88 or SEQ ID NO: 89. Suitably FR4 of the polypeptide of the invention comprises of more suitably consists of SEQ ID NO: 90.

Full Length Sequences Derived from 5G9

Suitably the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, with any one of SEQ ID NOs: 15 to 31, 43 or 44, more suitably SEQ ID NOs: 24-31, more suitably SEQ ID NO: 29 or SEQ ID NO: 30.

Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to any one of SEQ ID NOs: 15 to 31, 43 or 44, more suitably SEQ ID NOs: 24-31, more suitably SEQ ID NO: 29 or SEQ ID NO: 30. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to any one of SEQ ID NOs: 15 to 31, 43 or 44, more suitably SEQ ID NOs: 24-31, more suitably SEQ ID NO: 29 or SEQ ID NO: 30. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to any one of SEQ ID NOs: 15 to 31, 43 or 44, more suitably SEQ ID NOs: 24-31, more suitably SEQ ID NO: 29 or SEQ ID NO: 30.

Suitably the N-terminus of the polypeptide is D. Suitably the polypeptide comprises or more suitably consists of any one of SEQ ID NOs: 15 to 31, 43 or 44, more suitably SEQ ID NOs: 24-31, more suitably SEQ ID NO: 29 or SEQ ID NO: 30.

Full Length Sequences Derived from 7F6

Suitably the polypeptide of the present invention comprises or more suitably consists of a sequence sharing 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or greater sequence identity, with any one of SEQ ID NOs: 32 to 39, more suitably SEQ ID NOs: 33-38, more suitably SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, more suitably SEQ ID NO: 35.

Alternatively, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 addition(s) compared to SEQ ID NOs: 32 to 39, more suitably SEQ ID NOs: 33-38, more suitably SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, more suitably SEQ ID NO: 35. Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 substitution(s) compared to SEQ ID NOs: 32 to 39, more suitably SEQ ID NOs: 33-38, more suitably SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35, more suitably SEQ ID NO: 35.

Suitably, the polypeptide of the present invention comprises or more suitably consists of a sequence having no more than 20, more suitably no more than 15, more suitably no more than 10, more suitably no more than 9, more suitably no more than 8, more suitably no more than 7, more suitably no more than 6, more suitably no more than 5, more suitably no more than 4, more suitably no more than 3, more suitably no more than 2, more suitably no more than 1 deletion(s) compared to any one of SEQ ID NOs: 32 to 39, more suitably SEQ ID NOs: 33-38, more suitably SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35; more suitably SEQ ID NO: 35.

Suitably the N-terminus of the polypeptide is D. Suitably the polypeptide comprises or more suitably consists of any one of SEQ ID NOs: 32 to 39, more suitably SEQ ID NOs: 33-38, more suitably SEQ ID NO: 33, SEQ ID NO: 34 or SEQ ID NO: 35; more suitably SEQ ID NO: 35.

A comparison of the full length percentage identity and the number of identical residues between some of the polypeptides described herein is provided below. Polypeptides of the invention are highlighted in bold.

Full length percentage identity comparison and number of identical residues between ICVDs described herein:

Polynucleotide Sequences

Suitably, the polynucleotides used in the present invention are isolated. An “isolated” polynucleotide is one that is removed from its original environment. For example, a naturally-occurring polynucleotide is isolated if it is separated from some or all of the coexisting materials in the natural system. A polynucleotide is considered to be isolated if, for example, it is cloned into a vector that is not a part of its natural environment or if it is comprised within cDNA.

In one aspect of the invention there is provided a polynucleotide encoding the polypeptide or construct of the invention. Suitably the polynucleotide comprises or consists of a sequence sharing 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any one of SEQ ID NOs: 59 to 80. More suitably the polynucleotide comprises or consists of any one of SEQ ID NOs: 59 to 80. In a further aspect there is provided a cDNA comprising said polynucleotide.

In one aspect of the invention there is provided a polynucleotide comprising or consisting of a sequence sharing 70% or greater, such as 80% or greater, such as 90% or greater, such as 95% or greater, such as 99% or greater sequence identity with any one of the portions of any one of SEQ ID NOs: 59 to 80 which encodes CDR1, CDR2 or CDR3 of the encoded immunoglobulin chain variable domain.

Sequence Analysis

Sequence analysis as detailed in Example 17 has revealed that particularly preferred ICVDs of the invention contain one or more conserved differences from their germline equivalent sequence (i.e. maturated residues that, in particular ICVDs of the invention, are maintained during the maturation process, due to their contribution to binding cognate antigen). These conserved amino acids are highlighted in respect of the 5G9 and 7F6 sequences illustrated below (T18, T21, V33, G52, G56, F62 and Y93; according to Kabat numbering). The residues believed to be particularly important for maintenance of optimal potency are V33, G52, G56 and Y93.

Also highlighted below are insertion sequences NIN (H29A-H29C), NSL (H82A-H82C) and SPFN/SPHN (H100A-H100D.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein the immunoglobulin chain variable domain originates from a llama V gene which is either the Vo or Vq gene and from a llama J gene which is either the J6 or J6.1 gene and which differs from an immunoglobulin chain variable domain encoded by said genes by comprising one or more amino acids selected from T18, T21, V33, G52, G56, F62 and Y93 and conservative substitutions of said amino acids at these positions, according to Kabat numbering.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein the immunoglobulin chain variable domain originates from a llama V gene which is either the Vo or Vq gene and from a llama J gene which is either the J6 or J6.1 gene and which differs from an immunoglobulin chain variable domain encoded by said genes by comprising one or more amino acids selected from V33, G52, G56 and Y93, and optionally one or more amino acids selected from T18, T21 and F62, according to Kabat numbering.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and, wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from T18, T21, V33, G52, G56, F62 and Y93 and conservative substitutions of said amino acids at these positions, according to Kabat numbering.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and, wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from V33, G52, G56 and Y93, and optionally one or more amino acids selected from T18, T21 and F62, according to Kabat numbering.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from amino acids at positions which correspond to T18, T21, V36, G55, G59, F65 and Y99 of SEQ ID NO. 29 and conservative substitutions of the said amino acids at these positions.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from amino acids at positions which correspond to V36, G55, G59 and Y99 of SEQ ID NO. 29, and optionally one or more amino acids selected from amino acids at positions which correspond to T18, T21 and F65 of SEQ ID NO. 29.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from amino acids at positions which correspond to T18, T21, V33 G52, G56, F62 and Y96 of SEQ ID NO: 35 and conservative substitutions of the said amino acids at these positions.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence sharing 50% or greater sequence identity with SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from amino acids at positions which correspond to V33, G52, G56 and Y96 of SEQ ID NO: 35, and optionally one or more amino acids selected from amino acids at positions which correspond to T18, T21 and F62 of SEQ ID NO: 35.

Suitably the immunoglobulin chain variable domain comprises two or more, more suitably three or more, more suitably four or more, more suitably five or more, more suitably six or more, most suitably all of these amino acids, or conservative substitutions thereof, at these positions. More suitably still, the immunoglobulin chain variable domain comprises all of these amino acids.

Suitably CDR3 comprises a sequence sharing 55% or greater sequence identity with the sequences recited above, more suitably 65% or greater sequence identity or more suitably 75% or greater sequence identity.

Linkers and Multimers

A construct according to the invention comprises multiple polypeptides and therefore may suitably be multivalent. Such a construct may comprise at least two identical polypeptides according to the invention. A construct consisting of two identical polypeptides according to the invention is a “homobihead”. In one aspect of the invention there is provided a construct comprising two or more identical polypeptides of the invention.

Alternatively, a construct may comprise at least two polypeptides which are different, but are both still polypeptides according to the invention (a “heterobihead”).

Alternatively, such a construct may comprise (a) at least one polypeptide according to the invention and (b) at least one polypeptide such as an antibody or antigen-binding fragment thereof, which is not a polypeptide of the invention (also a “heterobihead”). The at least one polypeptide of (b) may bind IL-6R (for example via a different epitope to that of (a)), or alternatively may bind to a target other than IL-6R. Suitably the different polypeptide (b) binds to, for example, another pro inflammatory cytokine or chemokine or their respective receptors, other inflammatory mediators or immunologically relevant ligands involved in human pathological processes.

Constructs can be multivalent and/or multispecific. A multivalent construct (such as a bivalent construct) comprises two or more binding polypeptides therefore presents two or more sites at which attachment to one or more antigens can occur. An example of a multivalent construct could be a homobihead or a heterobihead. A multispecific construct (such as a bispecific construct) comprises two or more different binding polypeptides which present two or more sites at which either (a) attachment to two or more different antigens can occur or (b) attachment to two or more different epitopes on the same antigen can occur. An example of a multispecific construct could be a heterobihead. A multispecific construct is multivalent.

Suitably, the polypeptides comprised within the construct are antibody fragments. More suitably, the polypeptides comprised within the construct are selected from the list consisting of: a VHH, a VH, a VL, a V-NAR, a Fab fragment and a F(ab)′2 fragment. More suitably, the polypeptides comprised within the construct are VHHs.

The polypeptides of the invention can be linked to each other directly (i.e. without use of a linker) or via a linker. Suitably, the linker is a protease-labile or a non-protease-labile linker. The linker is suitably a polypeptide and will be selected so as to allow binding of the polypeptides to their epitopes. If used for therapeutic purposes, the linker is suitably non-immunogenic in the subject to which the polypeptides are administered. Suitably the polypeptides are all connected by non-protease-labile linkers. Suitably the non-protease-labile linkers are of the format (G₄S)_(x). Suitably x is 1 to 10, most suitably x is 6. Suitably the protease-labile linker is of the format [-(G_(a)S)_(x)—B-(G_(b)S)_(y)-]_(z) wherein a is 1 to 10; b is 1 to 10; x is 1 to 10; y is 1 to 10, z is 1 to 10 and B is K or R. Suitably a is 2 to 5, more suitably a is 4. Suitably b is 2 to 5, more suitably b is 4. Suitably x is 1 to 5, more suitably x is 1. Suitably y is 1 to 5, more suitably y is 1. Suitably z is 1 to 3, more suitably z is 1. Suitably B is K.

Vectors and Hosts

The term “vector”, as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g. non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions, and also bacteriophage and phagemid systems. The invention also relates to nucleotide sequences that encode polypeptide sequences or multivalent and/or multispecific constructs. The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell.

In one aspect of the invention there is provided a vector comprising the polynucleotide encoding the polypeptide or construct of the invention or cDNA comprising said polynucleotide. In a further aspect of the invention there is provided a host cell transformed with said vector, which is capable of expressing the polypeptide or construct of the invention. Suitably the host cell is a bacterium such as Escherichia coli a yeast or mould belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia, such as Saccharomyces cerevisiae or Pichia pastoris. Suitably the recombinant host is a yeast. Suitably the recombinant host is a mould. Suitably the yeast belongs to the genera Saccharomyces, Kluyveromyces, Hansenula or Pichia. Further examples of yeasts are those belonging to the genera Candida and Torulopsis. Suitably the mould belongs to the genus Aspergillus. Further examples of moulds are those belonging to the genera Acremonium, Altemaria, Chrysosporium, Cladosporium, Dictyostelium, Fusarium, Mucor, Penicillium, Rhizopus, Stachybotrys, Trichoderma and Trichophyton.

Stability

For the provision of an oral medicament, it is desirable to predict the stability of an ICVD in the intestinal tract of a model organism (such as mouse or cynomolgus monkey) and a target organism (such as a human). The activities of the major small intestinal proteases, trypsin and chymotrypsin, are conserved across mammalian species, whereas proteases present in the large intestine are likely to be produced by host species-specific gut microflora. To generate testing solutions (or testing ‘matrices’) that reflect these two environments, pooled mouse small intestinal supernatants and pooled human faecal supernatants may be prepared and used as outlined below. Both of these solutions are highly digestive towards unselected, un-engineered ICVDs.

Accordingly, suitable methods for determining the stability of an anti-IL6R agent in the intestinal tract include:

1A. The Standard Mouse Small Intestinal Supernatant Digestion Assay

An anti-IL6R agent can be assayed for stability in the intestinal tract by exposure to supernatant extract prepared from the pooled small intestinal contents of several mice. Following incubation of the anti-IL6R agent in the presence of the intestinal extract for a given time period, IL6R-binding activities of the “digested” samples are assayed using the anti-IL-6R gp130 ELISA provided below, and compared to the binding activity of “undigested” samples.

To produce mouse small intestinal supernatant, mouse small intestines are excised, the solid contents removed and the intestines rinsed with 1 ml of 0.9% saline. The solid and liquid parts are combined in 1.5 mL microfuge tubes, vortexed to homogeny and centrifuged at 16 k×rcf for 10 minutes at 4 degrees C. The supernatants are removed and stored at −80 degrees C. before being assayed further.

1B. The Standard Human Faecal Extract Digestion Assay

Alternatively, an anti-IL6R agent can be assayed for stability in the intestinal tract by exposure to human faecal supernatant prepared from pooled faecal supernatants of several humans. Following incubation of the anti-IL6R agent in the presence of the faecal supernatant for different time periods, IL6R-binding activities of the “digested” samples are assayed using the anti-IL-6R gp130 ELISA provided below, and compared to the binding activity of “undigested” samples.

To produce pooled human faecal supernatant, several faecal samples are turned into slurries with addition of 1×PBS. The slurries are then pooled, centrifuged and the supernatants removed, aliquoted and stored at −80 degrees C. This process removes the faecal matrix, including any cellular material.

2. The Standard Anti-IL-6R gp130 ELISA Performed on Samples Previously Exposed to Proteolytic Material

The objective of this assay is to measure the remaining concentration of active anti-IL-6R ICVDs which have previously been incubated in the presence of proteolytic material, such as mouse small intestinal supernatant or human faecal extract, thereby elucidating the impact on the ICVD of any proteolysis which may have taken place during incubation and therefore the proteolytic stability of the anti-IL-6R ICVDs. This assay detects binding of hIL-6R/hIL-6 complexes to recombinant human gp130. This interaction can be competitively inhibited by anti-IL-6R ICVDs, causing reduced binding of hIL-6R-hIL-6 complexes to gp130. Therefore, high signal in this ELISA represents a low concentration or low affinity of anti-IL-6R ICVD remaining active, and vice versa. The % survival is the percentage concentration of active ICVD, interpolated using the standard curve, maintained between a sample before and after digestion.

Materials Solutions Required: 1×PBS 1% BSA in PBS PBST (1×PBS, 0.05% Tween 20)

Block buffer (1% BSA in 1×PBS, pH 7.3-7.5) Assay buffer (1% BSA, 2× protease inhibitor* in 1×PBS) 0.5 M Sulphuric acid (H₂SO₄) *2× protease inhibitor=1 tablet per 50 ml buffer

Reagents Required:

Recombinant soluble human gp130 at known concentration SigmaFast protease inhibitor tablets (S8820) ICVD stock of known concentration (preferably 250 ug/mL diluted to 20 ug/mL in protease testing solution) Soluble human IL-6 at known concentration Soluble human IL-6R at known concentration Biotinylated goat anti-IL-6R polyclonal antibody (R&D systems BAF227); resuspended at 250 ug/ml in sterile PBS.

ExtrAvidin-Peroxidase (Sigma E2886)

TMB substrate (Microwell Peroxidase substrate System 2-C, KPL, 50-70-00)

Procedure Preparation:

-   -   1. Determine number of plates required for the assay.     -   2. Prepare the relevant volume (up to 3 plates at a time) of 0.2         μg/ml recombinant soluble human gp130 in PBS+5 μg/ml BSA.     -   3. Working quickly, dispense 50 μl/well into Maxisorp 96-well         ELISA plates (Nunc), loading a maximum of 4 plates in one batch.     -   4. Shake plate briefly, seal and incubate at 4° C. overnight.

Assay:

1. Wash the ELISA plate using a plate washer (4×˜380 μl PBST). Bang the plate on towel to remove residual liquid. 2. Apply 200 μl/well block buffer. Seal and incubate on a rotary plate shaker for ≥1 hour. 3. Prepare a dilution series of ICVD standards between 0.004 nM to 1000 nM in minimum final volumes of 70 μl using assay buffer as a diluent. 4. Prepare appropriate dilutions of samples to be tested in assay buffer, such that their estimated final concentration on the plate will fall in the range of 0.001 nM to 250 nM ICVD. Ensure that samples containing GI/faecal material are kept on ice as much as possible. 5. Prepare a 400 ng/ml IL-6 solution in assay buffer. 6. Prepare a 40 ng/ml IL-6R solution in assay buffer. 7. In a separate 96-well plate, mix together 50 μl of each ICVD dilution with 50 μl IL-6 solution. In each dilution series include one well with no ICVD. 8. In a further additional 96-well plate, mix together 85 μl ICVD-IL-6 mixture from step 7 with 85 μl IL-6R solution prepared in step 6. Include wells containing assay buffer only, such that the following controls are applied to each plate: IL-6 only, and no ICVD (IL6+IL6R only). Incubate for 5 minutes on rotary plate shaker. 9. Wash blocked ELISA plate as in step 1. 10. Transfer 50 μl of the mixtures prepared in step 8 to the washed ELISA plate in triplicate. Seal and incubate on a rotary plate shaker for 2 hours. 11. Wash blocked ELISA plate as in step 1. 12. Prepare 5 ml/plate 125 ng/mL of BAF227 anti-hIL-6R antibody made up in block buffer. Add 50 μl/well, seal, shake briefly, and incubate for 1 hour at room temperature or overnight at 4° C. 13. Wash blocked ELISA plate as in step 1. 14. Prepare 5 ml/plate 1/1000-1/3000 dilution of Extravidin in block buffer. Add 50 μl/well, seal, and incubate on a rotary shaker ≤30 mins 15. Wash blocked ELISA plate as in step 1. 16. Prepare 10 ml/plate TMB substrate (1:1 ratio of substrate A and B). Add 100 μl/well, seal and incubate on a rotary plate shaker until a mid blue colour evolves in the lowest dilution wells or up to a maximum of 30 mins. Shield from light. 17. Stop reaction with 50 μl/well 0.5 M H₂SO₄. 18. Read plate at 450 nm. 19. Use standard curve to interpolate concentrations of active ICVD. Raw OD450 values are adjusted with readings taken from blank control wells. Standard curves are plotted using appropriate software (e.g. Graphpad Prism using Log(inhibitor) vs. response-variable slope (four parameters)). ICVD concentrations in the test samples are calculated in the software using the standard curve. The active ICVD concentration in the test sample is expressed as a % of that in the 0 h sample to give % survival

Suitably the polypeptide or construct of the present invention retains 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% or more of the original potency of the polypeptide of the invention or construct after exposure to proteases present in the small and/or large intestine and/or IBD inflammatory proteases, such as after at least 6 hours', suitably after at least 16 hours' exposure to human faecal extract, for example in the standard human faecal extract assay.

Suitably the polypeptide or construct of the present invention retains 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% or more of the original potency of the polypeptide of the invention or construct after exposure to proteases present in the small and/or large intestine and/or IBD inflammatory proteases, such as after at least 1 hour's, suitably after at least 4 hours', suitably after at least 7 hours' exposure to mouse small intestinal supernatant, for example in the standard mouse small intestinal supernatant assay.

Suitably the polypeptide or construct of the present invention substantially retains neutralisation ability when exposed to proteases present in the small and/or large intestine and/or IBD inflammatory proteases when suitably 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% or more of the original potency of the polypeptide of the invention or construct is retained after exposure to proteases present in the small and/or large intestine and/or IBD inflammatory proteases, such as after at least 6 hours', suitably after at least 16 hours' exposure to human faecal extract, for example in the standard human faecal extract assay.

Suitably the polypeptide or construct of the present invention substantially retains neutralisation ability when exposed to proteases present in the small and/or large intestine and/or IBD inflammatory proteases when suitably 10%, more suitably 20%, more suitably 30%, more suitably 40%, more suitably 50%, more suitably 60%, more suitably 70%, more suitably 80%, more suitably 90%, more suitably 95%, more suitably 100% or more of the original potency of the polypeptide of the invention or construct is retained after exposure to proteases present in the small and/or large intestine and/or IBD inflammatory proteases, such as after at least 1 hour's, suitably after at least 4 hours', suitably after at least 7 hours' exposure to mouse small intestinal supernatant, for example in the standard mouse small intestinal supernatant assay.

Suitably 10% or more, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more of the administered dose of polypeptides or constructs of the invention retains neutralisation ability against IL-6R and remain in the faeces of a mouse, cynomolgus monkey and/or human (suitably excreted faeces or faeces removed from the intestinal tract) after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 hours of exposure to conditions of the intestinal tract.

A polypeptide of the invention or construct of the present invention remains substantially intact when suitably 10%, more suitably 20% or more, more suitably 30% or more, more suitably 40% or more, more suitably 50% or more, more suitably 60% or more, more suitably 70% or more, more suitably 80% or more, more suitably 90% or more, more suitably 95% or more, more suitably 99% or more, most suitably 100% of the administered quantity of polypeptide of the invention or construct remains intact after exposure to proteases present in the small and/or large intestine and/or IBD inflammatory proteases.

Suitably, the polypeptide or construct of the present invention substantially retains neutralisation ability and/or potency when delivered orally and after exposure to the intestinal tract (for example, after exposure to proteases of the small and/or large intestine and/or IBD inflammatory proteases). Such proteases include enteropeptidase, trypsin, chymotrypsin, and irritable bowel disease inflammatory proteases (such as MMP3, MMP12 and cathepsin).

Proteases of, or produced in, the small and/or large intestine include proteases sourced from intestinal commensal microflora and/or pathogenic bacteria, for example wherein the proteases are cell membrane-attached proteases, excreted proteases and proteases released on cell lysis). Most suitably the proteases are trypsin and chymotrypsin.

Suitably the intestinal tract is the intestinal tract of a dog, pig, human, monkey or mouse. Suitably the intestinal tract is the intestinal tract of a dog, pig, human, cynomolgus monkey or mouse. The small intestine suitably consists of the duodenum, jejunum and ileum. The large intestine suitably consists of the cecum, colon, rectum and anal canal. Suitably the polypeptide or construct of the invention is substantially resistant to one or more proteases. The intestinal tract, as opposed to the gastrointestinal tract, consists of only the small intestine and the large intestine.

Therapeutic Use and Delivery

A therapeutically effective amount of a polypeptide, pharmaceutical composition or construct of the invention, is an amount which is effective, upon single or multiple dose administration to a subject, in neutralising IL-6R to a significant extent in a subject. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the polypeptide, pharmaceutical composition or construct to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the polypeptide of the invention, pharmaceutical composition or construct are outweighed by the therapeutically beneficial effects. The polypeptide or construct of the invention can be incorporated into pharmaceutical compositions suitable for administration to a subject. The polypeptide or construct of the invention can be in the form of a pharmaceutically acceptable salt.

A pharmaceutical composition of the invention may suitably be formulated for oral, intramuscular, subcutaneous or intravenous delivery. The pharmaceutical compositions of the invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. Solid dosage forms are preferred. The polypeptide of the invention, pharmaceutical composition or construct may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

Typically, the pharmaceutical composition comprises a polypeptide or construct of the invention and a pharmaceutically acceptable diluent or carrier. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the polypeptide or construct of the invention. Pharmaceutical compositions may include antiadherents, binders, coatings, disintegrants, flavours, colours, lubricants, sorbents, preservatives, sweeteners, freeze dry excipients (including lyoprotectants) or compression aids.

Most suitably, the polypeptide of the invention, pharmaceutical composition or construct of the invention is administered orally. A key problem with oral delivery is ensuring that sufficient polypeptide, pharmaceutical composition or construct reaches the area of the intestinal tract where it is required. Factors which prevent a polypeptide, pharmaceutical composition or construct of the invention reaching the area of the intestinal tract where it is required include the presence of proteases in digestive secretions which may degrade a polypeptide, pharmaceutical composition or construct of the invention. Suitably, the polypeptide, pharmaceutical composition or construct of the invention are substantially stable in the presence of one or more of such proteases by virtue of the inherent properties of the polypeptide or construct itself. Suitably, the polypeptide or construct of the invention is lyophilised before being incorporated into a pharmaceutical composition.

A polypeptide of the invention may also be provided with an enteric coating. An enteric coating is a polymer barrier applied on oral medication which helps to protect the polypeptide from the low pH of the stomach. Materials used for enteric coatings include fatty acids, waxes, shellac, plastics, and plant fibers. Suitable enteric coating components include methyl acrylate-methacrylic acid copolymers, cellulose acetate succinate, hydroxy propyl methyl cellulose phthalate, hydroxy propyl methyl cellulose acetate succinate (hypromellose acetate succinate), polyvinyl acetate phthalate (PVAP), methyl methacrylate-methacrylic acid copolymers, sodium alginate and stearic acid. Suitable enteric coatings include pH-dependent release polymers. These are polymers which are insoluble at the highly acidic pH found in the stomach, but which dissolve rapidly at a less acidic pH. Thus, suitably, the enteric coating will not dissolve in the acidic juices of the stomach (pH ˜3), but will do so in the higher pH environment present in the small intestine (pH above 6) or in the colon (pH above 7.0). The pH-dependent release polymer is selected such that the polypeptide or construct of the invention will be released at about the time that the dosage reaches the small intestine, particularly the jejunum and ileum.

A polypeptide, construct or pharmaceutical composition of the invention can be formulated into preparations for injection by dissolving, suspending or emulsifying them in an aqueous or non-aqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilisers, isotonic agents, suspending agents, emulsifying agents, stabilisers and preservatives. Acceptable carriers, excipients and/or stabilisers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, omithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as polysorbates, POE ethers, poloxamers, Triton-X, or polyethylene glycol. Suitably the polypeptide of the invention is formulated and/or administered with a milk and bicarbonate mixture.

A pharmaceutical composition of the invention may be delivered topically to the skin (for example for use in the treatment of an autoimmune disease such as psoriasis or eczema). Such a pharmaceutical composition may suitably be in the form of a cream, ointment, lotion, gel, foam, transdermal patch, powder, paste or tincture and may suitably include vitamin D3 analogues (e.g calcipotriol and maxacalcitol), steroids (e.g. fluticasone propionate, betamethasone valerate and dobetasol propionate), retinoids (e.g. tazarotene), coal tar and dithranol. Topical medicaments are often used in combination with each other (e.g. a vitamin D3 and a steroid) or with further agents such as salicylic acid. If the pharmaceutical composition of the invention is to be delivered topically for the treatment of psoriasis or eczema, suitably a further substance considered to be effective in treating psoriasis or eczema may be included in the composition such as steroids especially Class 4 or Class 5 steroids such as hydrocortisone (eg 1% hydrocortisone cream); cyclosporin or similar macrolide agent or retinoids.

For all modes of delivery, the polypeptide, pharmaceutical composition or construct of the invention may be formulated in a buffer, in order to stabilise the pH of the composition, at a concentration between 5-50, or more suitably 15-40 or more suitably 25-30 g/litre. Examples of suitable buffer components include physiological salts such as sodium citrate and/or citric acid. Suitably buffers contain 100-200, more suitably 125-175 mM physiological salts such as sodium chloride. Suitably the buffer is selected to have a pKa close to the pH of the composition or the physiological pH of the patient.

Exemplary polypeptide or construct concentrations in a pharmaceutical composition may range from about 1 mg/mL to about 200 mg/ml or from about 50 mg/mL to about 200 mg/mL, or from about 150 mg/mL to about 200 mg/mL.

An aqueous formulation of the polypeptide, construct or pharmaceutical composition of the invention may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of suitable buffers include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, for example, on the buffer and the desired tonicity of the formulation.

The tonicity of the pharmaceutical composition may be altered by including a tonicity modifier. Such tonicity modifiers can be charged or uncharged chemical species. Typical uncharged tonicity modifiers include sugars or sugar alcohols or other polyols, preferably trehalose, sucrose, mannitol, glycerol, 1,2-propanediol, raffinose, sorbitol or lactitol (especially trehalose, mannitol, glycerol or 1,2-propanediol). Typical charged tonicity modifiers include salts such as a combination of sodium, potassium or calcium ions, with chloride, sulfate, carbonate, sulfite, nitrate, lactate, succinate, acetate or maleate ions (especially sodium chloride or sodium sulphate); or amino acids such as arginine or histidine. Suitably, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 1 mM to 500 nM. Suitably, at least one isotonic agent is included in the composition.

A surfactant may also be added to the pharmaceutical composition to reduce aggregation of the formulated polypeptide or construct and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Exemplary surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, and polysorbate 80. Exemplary concentrations of surfactant may range from about 0.001% to about 10% w/v.

A lyoprotectant may also be added in order to protect the polypeptide or construct of the invention against destabilizing conditions during the lyophilization process. For example, known lyoprotectants include sugars (including glucose, sucrose, mannose and trehalose); polyols (including mannitol, sorbitol and glycerol); and amino acids (including alanine, glycine and glutamic acid). Lyoprotectants can be included in an amount of about 10 mM to 500 mM.

The dosage ranges for administration of the polypeptide of the invention, pharmaceutical composition or construct of the invention are those to produce the desired therapeutic effect. The dosage range required depends on the precise nature of the polypeptide of the invention, pharmaceutical composition or construct, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patients condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.

Suitable daily dosages of polypeptide of the invention, pharmaceutical composition or construct of the invention are in the range of 50 ng-50 mg per kg, such as 50 ug-40 mg per kg, such as 5-30 mg per kg of body weight. The unit dosage can vary from less than 100 mg, but typically will be in the region of 250-2000 mg per dose, which may be administered daily or more frequently, for example 2, 3 or 4 times per day or less frequently for example every other day or once per week, once per fortnight or once per month.

In one aspect of the invention there is provided the use of the polypeptide, pharmaceutical composition or construct of the invention in the manufacture of a medicament for the treatment of autoimmune disease. In a further aspect of the invention there is provided a method of treating autoimmune disease comprising administering to a person in need thereof a therapeutically effective amount of the polypeptide, pharmaceutical composition or construct of the invention.

The word ‘treatment’ is intended to embrace prophylaxis as well as therapeutic treatment. Treatment of diseases also embraces treatment of exacerbations thereof and also embraces treatment of patients in remission from disease symptoms to prevent relapse of disease symptoms.

Combination Therapy

A pharmaceutical composition of the invention may also comprise one or more active agents (e.g. active agents suitable for treating the diseases mentioned herein). It is within the scope of the invention to use the pharmaceutical composition of the invention in therapeutic methods for the treatment of autoimmune diseases as an adjunct to, or in conjunction with, other established therapies normally used in the treatment of autoimmune diseases.

For the treatment of IBD (such as Crohn's disease or ulcerative colitis), possible combinations include combinations with, for example, one or more active agents selected from the list comprising: 5-aminosalicylic acid, or a prodrug thereof (such as sulfasalazine, olsalazine or bisalazide); corticosteroids (e.g. prednisolone, methylprednisolone, or budesonide); immunosuppressants (e.g. cyclosporin, tacrolimus, methotrexate, azathioprine or 6-mercaptopurine); anti-IL-6R antibodes (e.g. tocilizumab), anti-IL-6 antibodies, anti-TNF-alpha antibodies (e.g., infliximab, adalimumab, certolizumab pegol or golimumab); anti-IL12/IL23 antibodies (e.g., ustekinumab); anti-IL6R antibodies or small molecule IL12/IL23 inhibitors (e.g., apilimod); Anti-alpha-4-beta-7 antibodies (e.g., vedolizumab); MAdCAM-1 blockers (e.g., PF-00547659); antibodies against the cell adhesion molecule alpha-4-integrin (e.g., natalizumab); antibodies against the IL2 receptor alpha subunit (e.g., daclizumab or basiliximab); JAK3 inhibitors (e.g., tofacitinib or R348); Syk inhibitors and prodrugs thereof (e.g., fostamatinib and R-406); Phosphodiesterase-4 inhibitors (e.g., tetomilast); HMPL-004; probiotics; Dersalazine; semapimod/CPSI-2364; and protein kinase C inhibitors (e.g. AEB-071). The most suitable combination agents are tocilizumab, infliximab, adalimumab, certolizumab pegol or golimumab.

Hence another aspect of the invention provides a pharmaceutical composition of the invention in combination with one or more further active agents, for example one or more active agents described above.

In a further aspect of the invention, the polypeptide, pharmaceutical composition or construct is administered sequentially, simultaneously or separately with at least one active agent selected from the list above.

Similarly, another aspect of the invention provides a combination product comprising:

(A) a polypeptide, pharmaceutical composition or construct of the present invention; and (B) one or more other active agents, wherein each of components (A) and (B) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier. In this aspect of the invention, the combination product may be either a single (combination) formulation or a kit-of-parts. Thus, this aspect of the invention encompasses a combination formulation including a polypeptide, pharmaceutical composition or construct of the present invention and another therapeutic agent, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.

The invention also encompasses a kit of parts comprising components:

(i) a polypeptide, pharmaceutical composition or construct of the present invention in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier; and (ii) a formulation including one or more other active agents, in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier, which components (i) and (ii) are each provided in a form that is suitable for administration in conjunction with the other.

Component (i) of the kit of parts is thus component (A) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. Similarly, component (ii) is component (B) above in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier. The one or more other active agents (i.e. component (B) above) may be, for example, any of the agents mentioned above in connection with the treatment of autoimmune diseases such as IBD (e.g. Crohn's disease and/or ulcerative colitis). If component (B) is more than one further active agent, these further active agents can be formulated with each other or formulated with component (A) or they may be formulated separately. In one embodiment component (B) is one other therapeutic agent. In another embodiment component (B) is two other therapeutic agents. The combination product (either a combined preparation or kit-of-parts) of this aspect of the invention may be used in the treatment or prevention of an autoimmune disease (e.g. the autoimmune diseases mentioned herein).

Suitably the polypeptide, pharmaceutical composition or construct of the invention is for use as a medicament and more suitably for use in the treatment of an autoimmune and/or inflammatory disease.

Autoimmune Diseases and/or Inflammatory Diseases

Autoimmune diseases develop when the immune system responds adversely to normal body tissues. Autoimmune disorders may result in damage to body tissues, abnormal organ growth and/or changes in organ function. The disorder may affect only one organ or tissue type or may affect multiple organs and tissues. Organs and tissues commonly affected by autoimmune disorders include blood components such as red blood cells, blood vessels, connective tissues, endocrine glands such as the thyroid or pancreas, muscles, joints and skin. An inflammatory disease is a disease characterised by inflammation. Many inflammatory diseases are autoimmune diseases and vice-versa.

Autoimmune Diseases and/or Inflammatory Diseases of the GIT

The chronic inflammatory bowel diseases (IBD) Crohn's disease and ulcerative colitis, which afflict both children and adults, are examples of autoimmune and inflammatory diseases of the GIT (Hendrickson et al., 2002, herein incorporated by reference in its entirety). Ulcerative colitis is defined as a condition where the inflammatory response and morphologic changes remain confined to the colon. The rectum is involved in 95% of patients. Inflammation is largely limited to the mucosa and consists of continuous involvement of variable severity with ulceration, edema, and hemorrhage along the length of the colon (Hendrickson et al., 2002, herein incorporated by reference in its entirety). Ulcerative colitis is usually manifested by the presence of blood and mucus mixed with stool, along with lower abdominal cramping which is most severe during the passage of bowel movements. Clinically, the presence of diarrhoea with blood and mucus differentiates ulcerative colitis from irritable bowel syndrome, in which blood is absent. Unlike ulcerative colitis, the presentation of Crohn's disease is usually subtle, which leads to a later diagnosis. Factors such as the location, extent, and severity of involvement determine the extent of gastrointestinal symptoms. Patients who have ileocolonic involvement usually have postprandial abdominal pain, with tenderness in the right lower quadrant and an occasional inflammatory mass. Symptoms associated with gastroduodenal Crohn's disease include early satiety, nausea, emesis, epigastric pain, or dysphagia. Perianal disease is common, along with anal tags, deep anal fissures, and fistulae (Hendrickson et al., 2002, herein incorporated by reference in its entirety).

Crohn's disease and Ulcerative Colitis are primarily diseases of the intestinal tract and colon. Interleukin-6 is a proinflammatory cytokine that is considered to be involved in the pathogenesis of IBD and IL6-trans-signalling via soluble IL6-R is thought to play a particularly important role in mediating effects of IL-6 associated with the perpetuation of chronic intestinal inflammation (see Mitsuyama et al., 2007). High levels of IL-6 production have been found in cultured CD and UC intestinal mucosal biopsies (Gustot et al., 2005; Kusugami et al., 1995; Reimund et al., 1996; Hosokawa et al., 1999) associated with increased levels of sIL6R production (Hosokawa, 1999). Levels of IL-6 and sIL-6R production were greater in “involved” vs “non-involved” IBD tissue and were correlated with the severity of clinical disease. Lamina propria cells were found to be major producers of IL-6 (Reinecker et al., 1993; Kusugami et al., 1995; Reimund et al., 1996; Hosokawa et al., 1999) and sIL6Rs (Hosokawa et al., 1999). In cell cultures, IBD mucosal tissue-derived macrophages were the main cell type producing IL6 (Kusugami et al., 1995) and sIL6R (Hosokawa et al., 1999) on a per cell basis, while T cells, B cells and epithelial cells produced substantially lower amounts. Interestingly, the levels of IL6 production by IBD tissue and tissue derived cells far exceeded the levels of sIL6R production (see Hosokawa et al., 1999).

In these diseases the IL-6R is produced in the lamina propria underlying the gastrointestinal epithelium. This epithelium is disrupted in IBD and facilitates transport of the immunoglobulin chain variable domain into the lamina propria—the site of IL-6R production and biological action in these diseases (see Example 8). Other diseases of the GIT include for example the inflammatory disease mucositis (suitably drug- and radiation induced-mucositis) where inflammatory lesions are present in the mucosa disrupting the epithelial tight junctions which also allow the immunoglobulin chain variable domain access to the site of IL-6R production. In mucositis the lesions can occur anywhere from mouth to anus and for mouth and oesophagus lesions a mouthwash or cream preparation containing the variable domain may be used. For anal and rectal lesions, suppositories, creams or foams containing the variable domain would be suitable for topical application. The immunoglobulin chain variable domains will be cleared from the lamina propria or other inflammatory sites via absorption into the bloodstream at sites of inflammation or via lympatic clearance and subsequent entry into the bloodstream. The domains will therefore reach the liver via the bloodstream and will be cleared via glomerular filtration in the kidney. There is therefore good rationale that the domains will function therapeutically in diseases such as autoimmune hepatitis, type II diabetes and glomerular nephritis.

In inflamed IBD tissue, the production of IL-6 and shedding of IL-6Rs from activated macrophages results in the formation of soluble IL-6/sIL-6R complexes that can activate trans-signalling in cells that express only the gp130 subunit. This mechanism, which extends IL-6 responsiveness to an increased number of target cells, is considered to play an important role in the orchestration of mucosal inflammatory processes. The mechanism of IL-6 induced intestinal epithelial cell proliferation and regeneration is thought to involve signal transduction mediated via membrane bound IL-6 receptors (cis-signalling) (Rose-John, 2012; Waetzig & Rose-John 2012). The anti-human IL-6R antibody tocilizumab blocks both IL-6R classical signalling and IL-6/sIL-6R mediated trans-signalling and therefore blocks both pro-inflammatory, and potentially protective activities of IL-6. A rationale for the development of selective antagonists of IL-6-trans-signalling has been proposed based on the concept that this might avoid the inhibition of beneficial epithelial regenerative effects of IL-6 (see Rose-John et al, 2012; Waetzig & Rose-John 2012) that are mediated via mIL-6Rs (cis-signalling). An oral ICVD product with this profile could have safety and efficacy advantages over existing IL-6 neutralising antibodies for the treatment on Crohn's disease based on anti-inflammatory and improved mucosal healing properties.

Suitably the polypeptide, pharmaceutical composition or construct of the invention is used in the treatment of an autoimmune and/or inflammatory disease of the GI (gastrointestinal) tract where IL-6R (via, for example, the IL-6/IL-6R complex binding gp130) contributes to the pathology of such disease.

Suitably the polypeptide, pharmaceutical composition or construct of the invention is for use in the treatment of an autoimmune and/or inflammatory disease of the GI tract selected from the list consisting of Crohn's disease, ulcerative colitis, irritable bowel disease, diabetes type II, glomerulonephritis, autoimmune hepatitis, Sjogren's syndrome, celiac disease and drug- or radiation-induced mucositis (most suitably Crohn's disease).

Oral delivery of the immunoglobulin chain variable domain will ideally treat inflammatory diseases where IL-6R contributes to at least a proportion of the pathology and the immunoglobulin chain variable domain can access the tissue where the IL-6R is biologically active.

Autoimmune Diseases and/or Inflammatory Diseases of the Skin

The polypeptide of the invention may be incorporated into a cream/ointment or other topical carrier for administration to inflammatory skin lesions where IL-6R contributes to the pathology of such lesions.

Suitably the polypeptide, pharmaceutical composition or construct of the invention is for use in the treatment of an autoimmune and/or inflammatory disease of the skin selected from the list consisting of pemphigus, psoriasis, eczema, sderoderma, atopic dermatitis and fibrosis and inflammation induced cancers of the skin.

Suitably the polypeptide, pharmaceutical composition or construct is for use in the treatment of other autoimmune/inflammatory diseases in which IL-6R is responsible for a proportion of the pathology observed.

Preparative Methods

Polypeptides of the invention can be obtained and manipulated using the techniques disclosed for example in Green and Sambrook 2012.

Monoclonal antibodies can be produced using hybridoma technology, by fusing a specific antibody-producing B cell with a myeloma (B cell cancer) cell that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis (Köhler et al., 1975 and Nelson et al., 2000, herein incorporated by reference in their entirety). A monodonal antibody directed against a determined antigen can, for example, be obtained by:

a) immortalizing lymphocytes obtained from the peripheral blood of an animal previously immunized with a determined antigen, with an immortal cell and preferably with myeloma cells, in order to form a hybridoma, b) culturing the immortalized cells (hybridoma) formed and recovering the cells producing the antibodies having the desired specificity.

Alternatively, the use of a hybridoma cell is not required. Accordingly, monodonal antibodies can be obtained by a process comprising the steps of:

a) cloning into vectors, especially into phages and more particularly filamentous bacteriophages, DNA or cDNA sequences obtained from lymphocytes especially peripheral blood lymphocytes of an animal (suitably previously immunized with determined antigens), b) transforming prokaryotic cells with the above vectors in conditions allowing the production of the antibodies, c) selecting the antibodies by subjecting them to antigen-affinity selection, d) recovering the antibodies having the desired specificity.

Methods for immunizing camelids, cloning the VHH repertoire of B cells circulating in blood (Chomezynnski et al., 1987), and isolation of antigen-specific VHHs from immune (Arbabi-Ghahroudi et al., 1997) and nonimmune (Tanha et al 2002) libraries using phage, yeast, or ribosome display are known (WO92/01047, Nguyen et al., 2001 and Harmsen et al., 2007. These references are herein incorporated by reference in their entirety.

Antigen-binding fragments of antibodies such as the scFv and Fv fragments can be isolated and expressed in E. coli (Miethe et al., 2013, Skerra et al., 1988 and Ward et al., 1989, herein incorporated by reference in their entirety).

Mutations can be made to the DNA or cDNA that encode polypeptides which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli and S. cerevisiae, are known.

Mutation of polypeptides can be achieved for example by substitutions, additions or deletions to a nucleic acid encoding the polypeptide. The substitutions, additions or deletions to a nucleic acid encoding the polypeptide can be introduced by many methods, including for example error-prone PCR, shuffling, oligonucleotide-directed mutagenesis, assembly PCR, PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive ensemble mutagenesis, exponential ensemble mutagenesis, site-specific mutagenesis (Ling et al 1997, herein incorporated by reference in its entirety), gene reassembly, Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly (SLR) or a combination of these methods. The modifications, additions or deletions to a nucleic acid can also be introduced by a method comprising recombination, recursive sequence recombination, phosphothioate-modified DNA mutagenesis, uracil-containing template mutagenesis, gapped duplex mutagenesis, point mismatch repair mutagenesis, repair-deficient host strain mutagenesis, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, restriction-selection mutagenesis, restriction-purification mutagenesis, ensemble mutagenesis, chimeric nucleic acid multimer creation, or a combination thereof.

In particular, artificial gene synthesis may be used (Nambiar et al 1984, Sakamar et al., 1988, Wells et al., 1985 and Grundstrom et al., 1985, herein incorporated by reference in their entirety). A gene encoding a polypeptide of the invention can be synthetically produced by, for example, solid-phase DNA synthesis. Entire genes may be synthesized de novo, without the need for precursor template DNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. Products can be isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity (Verma et al., 1998)

Expression of immunoglobulin chain variable domains such as VHs and VHHs can be achieved using a suitable expression vector such as a prokaryotic cell such as bacteria, for example E. coli (for example according to the protocols disclosed in WO94/04678, which is incorporated herein by reference and detailed further below). Expression of immunoglobulin chain variable domains such as VHs and VHHs can also be achieved using eukaryotic cells, for example insect cells, CHO cells, Vero cells or suitably yeast or mould cells such as yeasts or moulds belonging to the genera Aspergillus, Saccharomyces, Kluyveromyces, Hansenula or Pichia. Suitably S. cerevisiae is used (for example according to the protocols disclosed in WO94/025591, which is incorporated herein by reference and detailed further below).

Specifically, VHHs can be prepared according to the methods disclosed in WO94/04678 using E. coli cells by a process comprising the steps of:

a) cloning in a Bluescript vector (Agilent Technologies) a DNA or cDNA sequence coding for the VHH (for example obtained from lymphocytes of camelids or produced synthetically) optionally including a His-tag, b) recovering the cloned fragment after amplification using a 5′ primer specific for the VHH containing an XhoI site and a 3′ primer containing the SpeI site having the sequence TC TTA ACT AGT GAG GAG ACG GTG ACC TG (SEQ ID NO: 13), c) cloning the recovered fragment in phase in the Immuno PBS vector (Huse et al., 1989, herein incorporated by reference in its entirety) after digestion of the vector with XhoI and SpeI restriction enzymes, d) transforming host cells, especially E. coli by transfection with the recombinant Immuno PBS vector of step c, e) recovering the expression product of the VHH coding sequence, for instance by affinity purification such as by chromatography on a column using Protein A, cation exchange, or a nickel-affinity resin if the VHH includes a His-tag.

Alternatively, immunoglobulin chain variable domains such as VHs and VHHs are obtainable by a process comprising the steps of:

a) obtaining a DNA or cDNA sequence coding for a VHH, having a determined specific antigen binding site, b) amplifying the obtained DNA or cDNA, using a 5′ primer containing an initiation codon and a HindIII site, and a 3′ primer containing a termination codon having a XhoI site, c) recombining the amplified DNA or cDNA into the HindIII (position 2650) and XhoI (position 4067) sites of a plasmid pMM984 (Merchlinsky et al., 1983, herein incorporated by reference in its entirety), d) transfecting permissive cells especially NB-E cells (Faisst et al., 1995, herein incorporated by reference in its entirety) with the recombinant plasmid, e) recovering the obtained products.

Further, immunoglobulin chain variable domains such as VHHs or VHs can be produced using E. coli or S. cerevisiae according to the methods disclosed in Frenken et al., 2000 and WO99/23221 (herein incorporated by reference in their entirety) as follows:

After taking a blood sample from an immunised llama and enriching the lymphocyte population via Ficoll (a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions—Pharmacia) discontinuous gradient centrifugation, isolating total RNA by acid guanidium thiocyanate extraction (Chomezynnski et al., 1987), and first strand cDNA synthesis (e.g. using a cDNA kit such as RPN 1266 (Amersham)), DNA fragments encoding VHH and VH fragments and part of the short or long hinge region are amplified by PCR using the specific primers detailed on pages 22 and 23 of WO99/23221. Upon digestion of the PCR fragments with PstI and HindIII or BstEII, the DNA fragments with a length between about 300 and 450 bp are purified via agarose gel electrophoresis and ligated in the E. coli phagemid vector pUR4536 or the episomal S. cerevisiae expression vector pUR4548, respectively. pUR4536 is derived from pHEN (Hoogenboom et al., 1991, herein incorporated by reference in its entirety) and contains the lacl gene and unique restriction sites to allow the cloning of the llama VHH and VH genes. pUR4548 is derived from pSY1 (Harmsen et al., 1993, herein incorporated by reference in its entirety). From this plasmid, the BstEII site in the leu2 gene is removed via PCR and the cloning sites between the SUC2 signal sequence and the terminator are replaced in order to facilitate the cloning of the VHNHH gene fragments. The VHNHHs have the c-myc tag at the C-terminus for detection. Individual E. coli JM109 colonies are transferred to 96 well microtiter plates containing 150 ml 2TY medium supplemented with 1% glucose and 100 mg L⁻¹ ampicillin. After overnight growth (37 degrees C.), the plates are duplicated in 2TY medium containing 100 mg L⁻¹ ampicillin and 0.1 mM IPTG. After another overnight incubation and optionally freezing and thawing, cells are centrifuged and pelleted and the supernatant can be used in an ELISA. Individual S. cerevisieae colonies are transferred to test tubes containing selective minimal medium (comprising 0.7% yeast nitrogen base, 2% glucose, supplemented with the essential amino acids and bases) and are grown for 48 h at 30 degrees C. Subsequently, the cultures are diluted ten times in YPGal medium (comprising 1% yeast extract, 2% bacto peptone and 5% galactose). After 24 and 48 h of growth, the cells are pelleted and the culture supernatant can be analysed in an ELISA. Absorbance at 600 nm (OD600) is optionally measured.

Further, immunoglobulin chain variable domains such as VH/VHHs can be produced using S. cerevisiae using the procedure as follows:

Isolate a naturally-occurring DNA sequence encoding the VH/VHH or obtain a synthetically produced DNA sequence encoding the VH/VHH, including a 5′-UTR, signal sequence, stop codons and flanked with SacI and HindIII sites (such a synthetic sequence can be produced as outlined above or for example may be ordered from a commercial supplier such as Geneart (Life Technologies)).

Use the restriction sites for transfer of the VH/VHH gene to the multi-copy integration (MCI) vector pUR8569 or pUR8542, as follows. Cut the DNA sequence encoding the VHH optionally contained within a shuttle vector, cassette or other synthetic gene construct and the MCI vector with SacI and HindIII using: 25 ul VHH DNA (Geneart plasmid or MCI vector), 1 ul SacI, 1 ul HindIII, 3 ul of a suitable buffer for double digestion such as NEB buffer 1 (New England Biolabs) overnight at 37 degrees C. Run 25 ul of digested DNA encoding the VHH and 25 ul of digested MCI vector on a 1.5% agarose gel with 1×TAE buffer and then perform gel extraction for example using QIAquick Gel Extraction Kit (Qiagen)). Set-up a ligation of digested MCI vector and digested DNA encoding the VHNHH as follows: 100 ng vector, 30 ng VHH gene, 1.5 ul 10× ligase buffer, 1 ul T4 DNA ligase, and ddH₂O. Then perform ligation overnight at 16 degrees C.

Next transform the E co/i cells. For chemical competent XL-1 blue cells, thaw 200 ul heat competent XL-1 blue cells and add 5 ul ligation mix on ice for about 30 minutes followed by heat shock for 90 seconds at 42 degrees C. Then add 800 ul Luria-Bertani low salt medium supplemented with 2% glucose and recover cells for 2 hours at 37 degrees C. Plate cells on Luria-Bertani agar and ampicillin (100 ug/ml) plates and keep overnight at 37 degrees C. For electro competent TG1 E. coli cells, use an electroporation cuvette. In the electroporation cuvette: thaw 50 ul electro competent TG1 cells and 1 ul ligation mix on ice for about 15 minutes. Place the cuvette in the holder and pulse. Add 500 ul of 2TY medium and recover cells for 30 minutes at 37 degrees C. Plate 100 ul of cells on Luria-Bertani, agar, containing ampicillin (100 ug/ml) and 2% glucose plates. Keep plates at 37 degrees C. overnight.

After cloning of the VHNHH gene into E. coli as detailed above, S. cerevisiae can be transformed with the linearized MCI vector. Before transformation is carried out, some steps are performed: (i) the DNA should be changed from circular to linear by digestion or else the DNA cannot be integrated into the yeast genome and (ii) the digested DNA should be cleaned of impurities by ethanol precipitation. Also, during the transformation process, the yeast cells are made semi-permeable so the DNA can pass the membrane.

Preparation for yeast transformation: perform a HpaI digestion of the midi-prep prepared from the selected E. coli colony expressing the VH/VHH gene as follows. Prepare a 100 ul solution containing 20 ng of midi-prep, 5 ul HpaI, 10 ul of appropriate buffer such as NEB4 buffer (BioLabs), and ddH₂O.

Cut the DNA with the HpaI at room temperature overnight. Next perform an ethanol precipitation (and put to one side a 5 ul sample from HpaI digestion). Add 300 ul ethanol 100% to 95 ul HpaI digested midiprep, vortex, and spin at full speed for 5 minutes. Carefully decant when a pellet is present, add 100 ul of ethanol 70%, then spin again for 5 minutes at full speed. Decant the sample again, and keep at 50-60 degrees C. until the pellet is dry. Re-suspend the pellet in 50 ul ddH₂O. Run 5 ul on a gel beside the 5 ul HpaI digested sample.

Yeast transformation: prepare YNBglu plates. Use 10 g agar+425 ml water (sterilised), 25 ml filtered 20×YNB (3.35 g YNB (yeast nitrogen base) in 25 ml sterilized H₂O) and 50 ml sterile 20% glucose and pour into petri dishes. Pick one yeast colony from the masterplate and grow in 3 ml YPD (Yeast Extract Peptone Dextrose) overnight at 30 degrees C. Next day prepare about 600 ml YPD and use to fill 3 flasks with 275 ml, 225 ml and 100 ml YPD. Add 27.5 ul yeast YPD culture to the first flask and mix gently. Take 75 ml from the first flask and put this in the second flask, mix gently. Take 100 ml from the second flask and put in the third one, mix gently. Grow until reaching an OD660 of between 1 and 2. Divide the flask reaching this OD over 4 Falcon tubes, ±45 ml in each. Spin for 2 minutes at 4200 rpm. Discard the supernatant. Dissolve the pellets in two Falcon tubes with 45 ml H₂O (reducing the number of tubes from 4 to 2). Spin for 2 minutes at 4200 rpm. Dissolve the pellets in 45 ml H₂O (from 2 tubes to 1). Spin for 2 minutes at 4200 rpm. Gently dissolve the pellets in 5 mL lithium acetate (LiAc) (100 mM), and spin for a few seconds. Carefully discard some LiAc, but retain over half of the LiAc in the tube. Vortex the cells, boil carrier DNA for 5 minutes and quickly chill in ice-water. Add to a 15 ml tube containing: 240 ul PEG, 50 ul cells, 36 uLiAc (1M), 25 ul carrier DNA, 45 ul ethanol precipitated VHNHH. Mix gently after each step (treat the blank sample the same, only without ethanol precipitated VHNHH). Incubate for 30 minutes at 30 degrees C., gently invert the tube 3-4 times, then heat shock for 20-25 minutes at 42 degrees C. Spin up to 6000 rpm for a brief time. Gently remove the supernatant and add 250 ul ddH₂O and mix. Streak all of it on an YNBglu plate until plates are dry and grow for 4-5 days at 30 degrees C. Finally, prepare YNBglu plates by dividing plates in 6 equal parts, number the parts 1 to 6, inoculate the biggest colony and streak out number 1. Repeat for other colonies from big to small from 1 to 6. Grow at 30 degrees C. for 3-4 days large until colonies are produced. The VH/VHH clones are grown using glucose as a carbon source, and induction of VH/VHH expression is done by turning on the Galactose-7-promoter by adding 0.5% galactose. Perform a 3 mL small scale culture to test the colonies and choose which one shows the best expression of the VH or VHH. This colony is then used in purification.

Purification: the VHNHH is purified by cation exchange chromatography with a strong anion resin (such as Capto S). On day 1, inoculate the selected yeast colony expressing the VHNHH in 5 ml YPD medium (YP medium+2% glucose) and grow the cells in 25 mL sealed sterile tubes at 30 degrees C. overnight (shaking at 180 rpm). On day 2, dilute the 5 ml overnight culture in 50 mL freshly prepared YP medium+2% glucose+0.5% galactose, grow the cells in 250 ml aerated baffled flasks at 30 degrees C. for two nights (shaking at 180 rpm). On day 4, spin the cells down in a centrifuge at 4200 rpm for 20 min. Cation exchange purification step using a strong anion resin: adjust the pH of the supernatant containing the ligand to 3.5. Wash 0.75 ml resin (+/−0.5 mL slurry) per of 50 mL supernatant with 50 mL of ddH₂O followed by three washes with binding buffer. Add the washed resin to the supernatant and incubate the suspension at 4 degrees C. on a shaker for 1.5 hours. Pellet the resin-bound VHNHH by centrifugation at 500 g for 2 minutes and wash it with wash buffer. Decant supernatant and re-suspend the resin with 10 mL of binding buffer. Put a filter in a PD-10 column, pour the resin in the column and let the resin settle for a while, then add a filter above the resin. Wait until all binding buffer has run through. Elute the VHNHH with 6×0.5 ml elution buffer. Collect the elution fractions in eppendorf tubes. Measure the protein concentration of the 6 eluted fractions with a Nanodrop. Pool the fractions that contain the VHH and transfer the solution into a 3,500 Da cutoff dialysis membrane. Dialyze the purified protein solution against 3 L of PBS overnight at 4 degrees C. On day 5, dialyze the purified protein solution against 2 L of fresh PBS for an additional 2 hours at 4 degrees C. Finally, calculate the final concentration by BCA.

Although discussed in the context of the V/VHH, the techniques described above could also be used for scFv, Fab, Fv and other antibody fragments if required.

Multiple antigen-binding fragments (suitably VHNHHs) can be fused by chemical cross-linking by reacting amino acid residues with an organic derivatising agent such as described by Blattler et al., 1985 (herein incorporated by reference in its entirety). Alternatively, the antigen-binding fragments may be fused genetically at the DNA level i.e. a polynucleotide construct formed which encodes the complete polypeptide construct comprising one or more antigen-binding fragments. One way of joining multiple antigen-binding fragments via the genetic route is by linking the antigen-binding fragment coding sequences either directly or via a peptide linker. For example, the carboxy-terminal end of the first antigen-binding fragment may be linked to the amino-terminal end of the next antigen-binding fragment. This linking mode can be extended in order to link antigen-binding fragments for the construction of tri-, tetra-, etc. functional constructs. A method for producing multivalent (such as bivalent) VHH polypeptide constructs is disclosed in WO 96/34103 (herein incorporated by reference in its entirety).

Suitably, the polypeptide of the invention (in particular, a VHH of the invention) can be produced in a fungus such as a yeast (for example, S. cerevisiae) comprising growth of the fungus on a medium comprising a carbon source wherein 50-100 wt % of said carbon source is ethanol, according to the methods disclosed in WO02/48382. Large scale production of VHH fragments in S. cerevisiae is described in Thomassen et al., 2002 (herein incorporated by reference in its entirety).

In one aspect of the invention there is provided a process for the preparation of the polypeptide or construct of the invention comprising the following steps:

i) cloning into a vector, such as a plasmid, the polynucleotide of the invention, ii) transforming a cell, such as a bacterial cell or a yeast cell capable of producing the polypeptide or construct of the invention, with said vector in conditions allowing the production of the polypeptide or construct, iii) recovering the polypeptide or construct, such as by affinity chromatography.

In one aspect of the invention there is provided a polypeptide comprising an immunoglobulin chain variable domain comprising three complementarity determining regions (CDR1-CDR3) and four framework regions; wherein the polypeptide has increased intestinal stability and/or potency relative to a corresponding polypeptide not having said insert, wherein FR1 comprises an insert of 4 to 5 amino acids, suitably 3 amino acids. Suitably the insert is the sequence NIN or three consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NIN. Most suitably FR1 comprises the sequence NIN. More suitably the last four C-terminal residues of FR1 are NINX or four consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NINX, wherein X is any amino acid. Suitably X is S or a conservative substitution of S, most suitably X is S. More suitably FR1 consists of 33 residues and the last four residues of FR1 are NINS.

In one aspect of the invention there is provided a method of increasing the intestinal stability of a polypeptide comprising an immunoglobulin chain variable domain, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions, wherein the method comprises the step of inserting into FR1 an insert of 4 to 5 amino acids, suitably 3 amino acids. Suitably the insert is the sequence NIN or three consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NIN. Most suitably FR1 comprises the sequence NIN. More suitably the last four C-terminal residues of FR1 are NINX or four consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NINX, wherein X is any amino acid. Suitably X is S or a conservative substitution of S, most suitably X is S. More suitably FR1 consists of 33 residues and the last four residues of FR1 are NINS.

In one aspect of the invention there is provided a method of making a polypeptide comprising an immunoglobulin chain variable domain, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions; wherein the polypeptide has increased intestinal stability relative to a corresponding polypeptide not having said insertions, wherein the method comprises the step of inserting into FR1 an insert of 4 to 5 amino acids, suitably 3 amino acids. Suitably the insert is the sequence NIN or three consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NIN. Most suitably FR1 comprises the sequence NIN. More suitably the last four C-terminal residues of FR1 are NINX or four consecutive amino acids wherein one or more of the amino acids is a conservative substitution of the corresponding amino acid in the sequence NINX, wherein X is any amino acid. Suitably X is S or a conservative substitution of S, most suitably X is S. More suitably FR1 consists of 33 residues and the last four residues of FR1 are NINS The present invention will now be further described by means of the following non-limiting examples.

EXAMPLES Example 1: Llama Immunisation, Phage Display and Immunoglobulin Chain Variable Domain Selection Immunisation

Two llamas (llama numbers 37 and 46) were each immunised with soluble human recombinant IL-6R, using two protocols to increase the chance of generating a range of ICVDs with significant sequence diversity and activity against the IL-6R. Blood cells collected from each llama at the end of each immunisation phase were used to generate six separate phage display libraries: one from each llama after the first round of immunisations and two from each llama after the second round of immunisations.

Immunisation protocol 1

The llamas received an initial prime with 50 ug soluble human recombinant IL-6R (shIL-6R) mixed 1:1 with stimmune adjuvant on day 0. The llamas were boosted with 50 ug of the same at days 14, and 28, and with 40 ug on day 35. All injections were intra-muscular. Blood was drawn on day 44 for the isolation of peripheral blood lymphocytes and extraction of RNA for library construction.

Immunisation Protocol 2

After a resting period of 11 weeks after the first protocol, each llama received two more boosts of 50 ug shIL-6R one week apart and with blood drawn 9 days later. Serum immune responses to immunisations were assessed for each of the llamas at several time-points by measuring the binding of heavy chain antibodies to immobilised human IL-6R using a plate ELISA format and detection with rabbit anti-heavy chain and donkey anti-rabbit IgG coupled to horseradish peroxidase (HRP). Titration curves obtained showed that each of the llamas had responded to the first round of immunisations giving mid-range titres of IL-6R-binding IgG heavy chain antibodies. The serum antibody titres did not increase following the second round of immunisations.

Construction of Phage Libraries

Blood cells collected from each llama at the end of each immunisation phase were used to generate six separate phage display libraries. Total RNA was extracted from the peripheral blood lymphocytes that were isolated from each of the immunised llamas (llamas 37 and 46). The RNA was then used to generate cDNA and PCR was performed to amplify specifically the variable regions of antibodies. cDNA fragments encoding the heavy chain repertoire were cloned into a phagemid vector and the library introduced into E. coli. The phage libraries were produced by culturing the E. coli with helper phage, and precipitation of the resulting heavy chain-displaying phage. The numbers in each library were determined by titration and infection of log-phase E. coli strain TG1 with the different dilutions. The libraries were estimated to contain 1×10⁸ unique heavy chain sequences.

Libraries 37-2 and 46-2

These libraries are from PMBC harvested after the first immunisations. The PCR primers used amplified VH3 family heavy chains. ICVD sequences in this library were c-terminally fused to Flag and 6-His affinity tags.

Libraries 37-3/3r and 46-3/3r

These libraries are from PMBCs harvested after the second immunisations. The PCR primers used amplified VH3 and VH4 family heavy chains. The ICVD sequences in libraries 37-3 and 46-3 were c-terminally fused to Flag and 6-His affinity tags, whereas in libraries 37-3r and 46-3r were c-terminally fused to only 6-His affinity tags.

Library Selections for Phage with Human IL6R-Binding Activity

Library selection strategies were established to isolate ICVDs that bind to epitopes present on the extracellular domain of the IL-6R alpha subunit including ICVDs that interfere either with (i) the binding of IL-6 to sIL-6R or (ii) with the association of IL-6-IL-6R complexes with gp130 subunits. Complexes formed between IL-6 and soluble (sIL-6R) or membrane (mIL-6R) IL-6 receptor subunits can associate with gp130 to form trans- or cis-IL-6R signalling complexes, respectively. A class of ICVD that block the interaction of IL-6 with the receptor binding site would be expected to inhibit formation of both trans- and cis-IL-6R signalling complexes. ICVD that target other regions of the IL-6R involved in receptor complex formation and that are non-competitive with IL-6 binding may be more efficient (and effective) for inhibition of the IL-6 pathway at sites of inflammation. Furthermore, ICVDs capable of selectively binding to either IL-6-sIL-6R or IL-6-mIL-6R complexes may preferentially antagonise either trans- or cis-IL-6R signalling. Accordingly, several methods were used for the selective enrichment of phages displaying ICVDs with the binding characteristics described above. These methods included the following:

1) Ability to inhibit IL-6R-IL6-gp130 complex formation: Methods for the library selection were chosen that would enrich for ICVDs that are capable of binding to IL-6R at the IL-6 binding site because ICVDs with these properties are most likely to block biological activity of IL-6R. Antibodies capable of inhibiting the complex formation have previously been shown to block a range of biological effects of IL-6R. For example, tocilizumab is a clinically-effective, humanised mouse mAb that binds IL-6R at the IL-6-binding site with high affinity (Kishimoto T. et al. 2006). High concentrations of a tocilizumab Fab fragment or IL-6 were used for the selective elution of this particular group of ICVDs. 2) Ability to bind IL-6R a) in solution and b) when bound to IL-6: A method for selection was developed for ICVDs that bind to the natural epitopes of IL-6R in solution (instead of IL-6R adsorbed to a plastic substrate) in the presence and absence of IL-6. Selections on soluble antigen may isolate ICVDs unrelated to those binding surface-adsorbed antigen. In this method, biotinylated antigen is mixed with phage and the phage-antigen complex is pulled down using avidin. This format also allows pre-incubation of IL-6 with the biotinylated shIL-6R to create a complex. 3) Desirable binding kinetics: high-affinity ICVDs have a low K_(d), which is a ratio of their speed of antigen-binding (K_(on)) and their slow rate of release (K_(off)). The length of the phage absorptions were decreased to select for a fast K_(on), and both the number of washes and the length of incubations were increased to select for a slow K_(on). 4) Improved stability against gastro-intestinal supernatants: the removal of unstable ICVDs from the phage libraries by digestion may improve the efficiency of the ICVD screening process. Proteolytic digestion of phage libraries has previously been used to select for stabilised proteins. These previous methods used low concentrations of protease due to the instability of the phage pill fusion protein. A new phagemid was developed showing increased protease stability of the pill fusion protein and used in short digestions with high concentrations of proteases or GI extracts. First Round Library Selections of Phage Displaying ICVDs with Human IL-6R-Binding Activity

Phage libraries were derived from the llamas after the first round immunisation (Library 37-2 and 46-2); a first selection was performed to isolate phage displaying ICVDs with IL-6R-binding activity by panning on human IL-6R. Soluble recombinant human IL-6R (1000 or 500 ng) was coated directly onto wells of a Maxisorp plate and phages were allowed to attach. After extensive washing, bound phages were collected using either (i) non-selective elution by alkaline pH shock using TEA or (ii) by selective displacement of ICVD by 1 ug IL-6. Phage from the 1000 ng first round selections were selected further on 100 or 10 ng IL-6R with the same washing and elution conditions as before.

Second Round Library Selections of Phage Displaying ICVDs with Human IL-6R-Binding Activity

Phage libraries were derived from the llamas after 2^(nd) round immunisation (Libraries 37-3 and 46-3). In the first instance, libraries were selected on 200 or 40 ng shIL-6R coated directly onto wells of a maxisorp plate, subjected to multiple washes, prolonged incubations with 1× PBS, and elution with either IL-6, tocilizumab Fab fragment or TEA.

ICVD 4D3 from the llama 46-3 library was selected at this stage by capture on 200 ng shIL-6R, incubated for 2 h with 1 ug/mL IL-6 and then eluted in a 24 h incubation with 1 ug/mL tocilizumab Fab fragment. In between the incubation and elution step, the plate was washed 10× with 1×PBS, 0.05% Tween20.

Phage from the 200 ng wells were then selected further on 200 and 20 ng IL-6R-coated wells, washed and eluted in similar conditions. In the initial selection, phage captured on 200 ng shIL-6R were eluted in a 2 hour incubation with the 1 ug/mL IL-6, bulked up and put through a secondary selection by capture on 200 ng IL6R and a 6 h elution with 1 ug/mL IL6. The phage were washed 10× with 1×PBS, 0.05% Tween20 in between the capture and elution steps in both elutions. ICVDs 5G9 and 3C12 were isolated from the llama 37-3 library at this stage.

A second set of selections on soluble, biotinylated IL-6R were performed to isolate phage against epitopes on the soluble form of IL-6R when in complex with IL-6. The libraries were mixed with 200, 40 or 8 ng of sIL-6R or sIL-6R in complex with IL-6. ICVD 7F6 was isolated from the llama 37-3 here after capture on 40 ng biotinylated shIL-6R and elution with TEA. The phage were washed 10× with 1×PBS, 0.05% Tween20 in between the capture and elution. The sIL-6R and bound phage were captured on neutravidin coated microplates, and the plates washed prior to elution using TEA. The phage from 200 ng selections went through another selection in identical conditions.

Third Round Library Selections of Phage Displaying ICVDs with Human IL-6R-Binding Activity

Phage Libraries from the llamas after 2nd round immunisation (Libraries 37-3r and 46-3r) were cloned into a newly developed phagemid that produces more protease-stable phage. The libraries were digested with either 1 mg/ml trypsin, 1 mg/mL trypsin and chymotrypsin or 20% rat GI extract before capturing the phage on 500 or 50 ng sIL6R-coated microwell plates. After the washing with 1×PBS, the phage were eluted in TEA. The rat GI digested phage captured on 500 ng IL-6R were further selected identical conditions after digestion in 20% rat GI extract.

Example 2: Generation and Screening of Periplasmic Extracts for Primary Evaluation

Phages present in eluates from the selections were used to infect E. coli TG1 cells. Colonies were picked randomly into twelve 96-well master plates (76 clones and 88 clones from Libraries 37-2 and 46-2, respectively into master plates VIL-1 and VIL-2; 396 clones and 340 clones from Libraries 37-3 and 46-3, respectively into master plates VIL-3 to VIL-10; 92 clones each from Libraries 37-3r and 46-3r into masterplates VIL-14 and VIL-15) and propagated to generate clonal cultures. Bacterial cell outer membranes were lysed by freeze-thawing to release the ICVD-containing periplasmic fraction. Cell debris was removed by centrifugation and the periplasmic supernatants containing the selected monoclonal ICVDs were transferred to fresh 96-well plates and used for further primary evaluation studies to identify those ICVDs with the required characteristics.

Phages present in eluates from the different library selections were used to infect E. coli and individual colonies were picked into master-plates and propagated to generate clonal cultures. Periplasmic supernatants containing the selected monoclonal ICVD were used for primary evaluation studies to identify those with the required characteristics.

The neutralising activity of anti-IL-6R ICVDs was measured in a plate ELISA format as the ability to inhibit binding of the IL-6-sIL-6R complex to gp130, either by interference with IL-6 binding to the IL-6R or by inhibition of the IL-6-sIL-6R interaction with gp130.

ICVDs were also tested for intrinsic resistance to inactivation in the presence of small intestinal proteases. Following protease digestions, ICVDs showing residual activity in the gp130-based plate ELISA were identified.

Cross-reactivity of ICVDs with cynomolgus monkey IL-6R was assessed using the gp130-based plate ELISA assay format but with cynomolgus monkey serum as a source of monkey sIL-6R. ICVDs inhibiting the formation of a cyno-IL-6R complex with human IL-6 and human gp130 were identified.

Amino acid sequences of selected ICVDs were determined and aligned to assess diversity and family relationships. From a total of 900 library-selected clones picked into the original 12 master-plates a final set of primary clones was selected for production in E. coli and the ICVDs affinity purified for more detailed evaluation studies.

These primary clones included ICVDs 7F6, 3C12, 4D3 and 5G9. Sequencing revealed that 5G9 and 7F6 shared similar CDR3s. 3C12 and 4D3 had CDR3s distinct from each other and distinct from the 5G9 and 7F6 ‘family’. The remaining selected primary clones also had CDR3s distinct from the 5G9 and 7F6 ‘family’. These remaining selected primary clones are not discussed further. ICVDs sharing similar CDR3s to the 5G9 and 7F6 ‘family’, and which are therefore polypeptides of the invention, are highlighted in bold in the tables below.

Example 3: Family Expansion to Produce 21E6

After the initial rounds of selecting primary clones, a family expansion approach was used (see Koh et al., 2010). Family expansion is a molecular biology technique that enriches for groups of related antibodies in display libraries. When these new libraries are subjected to selections and screens, a higher proportion of eluted antibodies will be related to the original clone, allowing for better sampling of the sequence diversity within a single family. In this case, a family expansion approach was therefore attempted to identify antibodies with similarly favourable antigen-binding characteristics to 7F6.

The family-expanded library was panned to capture the phage on IL6R, followed by a brief two hour elution with a competing protein (tocilizumab Fab/IL6) and then a long (16 elution) with the same proteins. Masterplates were picked from the selected clones, which then were digested with 1 mg/mL trypsin for 6 hours. The resulting protease resistant, potent clones were patched into a new masterplate and then reanalysed with 1 mg/mL trypsin+1 mg/mL chymotrypsin for 6 hours. Most ICVDs did not survive this treatment, but one ICVD which had not already been encountered appeared to survive better than the rest. This was 21E6. 21E6 is related to 7F6 and differs by two mutations: Q13L and F106L. 21E6 was expressed from a phagemid vector and purified for testing in digest and potency assays.

Example 4: Evaluation of the Potency and Maximal IL-6R Neutralising Activity of Purified E. coli Recombinant ICVDs

Production of Selected ICVD Clones in E. coli

Selected ICVDs were re-cloned into the vector pMEK222 (thus introducing C-terminal Flag and 6×His tags) for production in E. coli and the ICVDs affinity purified for more detailed evaluation studies.

IL-6R-neutralising activities of the purified ICVDs were evaluated in several in vitro assay systems to assess potency and efficacy against soluble and membrane forms of human IL-6R and soluble cynomolgus monkey IL-6R.

Inhibition of gp130/IL-6/IL-6R Complex Formation—Potency in Neutralising Soluble Human IL6R

The standard gp130 ELISA assay detailed above under ‘Potency, inhibition and neutralisation’ (referred to as ‘the gp130 ELISA’ hereon) was performed with the modifications described below to test the ability of anti-IL-6R ICVDs to neutralise sIL-6R-IL-6 binding to gp130, demonstrating the potency of the recombinant purified ICVDs. Briefly, Maxisorp 96-well plates were coated overnight with 50 μl/well 0.5 μg/ml gp130 then blocked. ICVDs were serially diluted and mixed 1:1 with 100 ng/ml recombinant human IL-6 then 1:1 with 40 ng/ml recombinant human sIL-6R, and incubated for 1 hour to allow binding before adding to the gp130-coated plates. Bound IL-6R was detected with 50 μl/well 0.250 μg/ml BAF227 and then 50 μl/well 1/1000 diluted Extravidin-HRP and the level of neutralisation by the ICVD of IL-6R binding to IL-6/gp130 was determined.

Dose response curves were obtained (dose response curves of particular ICVDs illustrated in FIG. 1). In particular, 7F6 and 5G9 appeared to be substantially more potent than tocilizumab. 3C12 was not more potent than toclizumab. This was confirmed with curve-fitting software (Graphpad Prism), which fitted 4 parameter curves to the data and calculated the IC50s (Table 4.1, below).

TABLE 4.1 IC50s of particular ICVDs and tocilizumab in the first assay with 20 ng/mL IL6R and 100 ng/mL IL6 ICVD Name 7F6 3C12 4D3 5G9 tocilizumab IC50 (nM) 0.11 1.89 0.66 0.16 0.97

7F6 and 5G9 were more potent than 3C12, 4D3 or tocilizumab in this assay.

A separate assay was performed on 21E6 and 7F6 using the same protocol as above with the exception that the ICVD/IL-6R/IL-6 where only incubated with shaking for 10 min before being added to the assay plates. The results of the assay are shown in FIG. 2 and Table 4.2. It was found that 21 E6 was approximately two-fold less potent than 7F6.

TABLE 4.2 IC50s of 7F6 and 21E6 in the second assay with 20 ng/mL IL6R and 100 ng/mL IL6 ICVD Name 7F6 21E6 IC50 (nM) 0.16 0.3

In summary, highly potent ICVDs 7F6, 5G9 and 21E6, were identified. These ICVDs all share similar CDR3 sequences.

Cynomolgus Monkey sIL6R Cross-Reactivity

Cross-reactivity of anti-human IL6R ICVDs with cynomolgus monkey IL-6R would facilitate preclinical studies in this species. The cross-reactivity of the selected ICVDs was therefore investigated.

The standard anti-cynomolgus monkey IL-6R gp130 ELISA assay detailed under ‘Potency, neutralisation and inhibition’ above was performed to assay the ICVDs in parallel for their ability to neutralise sIL6R in cynomolgous monkey serum and recombinant purified human sIL6R. Briefly, Maxisorp 96-well plates were coated overnight with 50 μl/well 1 μg/ml gp130 then blocked. ICVDs were serially diluted and mixed 1:1 with 200 ng/ml recombinant human IL-6, then 1:1 with either 20 ng/ml recombinant human sIL-6R or a 5-fold dilution of cynomolgus monkey serum and incubated for 1 hour to allow binding before adding to the gp130-coated plates. Bound cynomolgus monkey IL-6R was detected with 50 μl/well either 0.25 μg/ml (for human IL-6R) or 0.5 μg/ml (for monkey serum) BAF227 and then 50 μl/well 1/1000 Extravidin-HRP and the level of neutralisation by the ICVD of IL-6R binding to IL-6/gp130 was determined

(dose response curves of particular ICVDs in inhibiting human gp130/IL-6/IL-6R interaction are illustrated in FIG. 3 and dose response curves of particular ICVDs in inhibiting human gp130/human IL-6/cynomolgous monkey IL-6R interaction are illustrated in FIG. 4).

To demonstrate the relative potencies of the ICVDs on the two sIL-6Rs, the IC50s were calculated as before, and the two IC50s were expressed as a ratio (i.e. how many fold more potent the ICVDs are in neutralising cynomolgous IL6R compared to human IL-6R. These ratios are shown in Table 4.3 below. In this iteration of the assay, 10 ng/mL human IL-6R was compared to 10× diluted cynomolgous serum in the presence of 50 ng/mL human IL-6.

Prior art anti-IL-6R polypeptide 20A11 was also included in this assay. 20A11 is disclosed in WO 2010/115998.

TABLE 4.3 Human and cynomolgous sIL-6R IC50 ratios of the ICVDs (human IC50 divided by cynomolgous IC50) Ratio Cyno IL6R Human IL6R Cyno IC50/ ICVD Name IC50 (nM) IC50 (nM) human IC50 3C12 4.142 3.531 0.9 4D3 0.372 0.6907 1.9 7F6 0.342 0.4243 1.2 5G9 0.8038 0.1477 0.2 20A11 0.1058 0.2401 2.3 (prior art comparative example) tocilizumab Fab 1.131275 0.7044 0.6 (prior art comparative example)

7F6 and 5G9 were found to have cross-reactivity with cynomolgous sIL-6R.

Inhibition of Membrane-IL-6R Induced Cell Proliferation in the TF1 Assay

Selected ICVDs were tested in the TF-1 assay as detailed above under ‘Potency, inhibition and neutralisation’; ‘The standard TF-1 cell assay’. IL-6 stimulates the proliferation of human TF-1 human erythroleukemic cells via the activation of membrane IL-6 receptors. The clinical anti-IL-6R monoclonal antibody tocilizumab potently and effectively inhibits IL-6-indiced TF-1 cell proliferation by interfering with the binding of IL-6 to mIL-6Rs on the cells. Selected ICVD were tested in this assay to compare their potencies and maximal inhibitory effects on IL-6 induced proliferation with a Fab fragment of the clinical standard tocilizumab.

Briefly, TF1 cells were grown to log phase in Low-serum RPMI 1640+2 nM L-glu+pen/strep+5% FCS+2.5 ug/mL GM-CSF. The cells were washed 2× in prewarmed 1×PBS, and plated at 2×10⁴ cells/well in 50 ul in 96 wells Costar micro-plates in assay medium (AM; Low-serum RPMI 1640+2 mM L-glu+Pen/Strep with no serum and no GM-CSF). The serial dilutions of anti-IL-6R ICVDs were prepared in AM at 4 times the assay concentration and mixed 1:1 with IL6 at 20 ng/mL (4 times the assay concentration) to have a 2× final concentration of ICVD and IL6. 50 uL of each solution were added to the cells. Plates were stored for 48 hours at 37° C. and 5% CO₂. The assay was read with 10 uL of resazurin and the cells were incubated for 2 h at 37° C. The assay was stopped with 50 uL of 3% SDS and the plates were then read with excitation at 540 nm and emission at 590 nm.

FIG. 5 shows the inhibition of IL6-induced proliferation of TF1 cells following incubation with increasing concentration of anti-IL-6R ICVD (in the figure, VIL-7F6 refers to 7F6, VIL-5G9 refers to 5G9, VIL-4D3 refers to 4D3, VIL-3C12 refers to 3C12 and PMP20A11 refers to 20A11). A Prism GraphPad analysis using 4 parameter non-linear regression curve provided the IC50 values in Table 4.4. All of these tested ICVDs exhibited a complete inhibition of TF1 proliferation at the highest concentration tested or lower (16-250 nM).

TABLE 4.4 IC50 values of ICVDs in TF1 assay Anti-IL-6R ICVD IC₅₀ (nM) 7F6 0.5 5G9 0.9 4D3 26.0 3C12 44.6 tocilizumab Fab 15.9 (prior art comparative example) 20A11 1.1 (prior art comparative example)

It can be seen that 7F6 and 5G9 are the most potent ICVDs in this assay with IC50 values of 0.5 nM and 0.9 nM respectively, which are lower than the tocilizumab Fab fragment, comparative example 20A11, 4D3 and 3C12.

A separate assay on 7F6 and 21 E6 was performed using a similar protocol to that used above. In this assay, low level GM-SCF contamination increased the background assay signal. Regardless, dose response curves were calculable. The fluorescence measured at different ICVD concentrations was plotted (see FIG. 6), inhibition curves were produced (not shown) and IC50 values obtained as described above. The results are shown in Table 4.5.

TABLE 4.5 IC50 values of 7F6 and 21E6 in TF1 assay Anti-IL-6R ICVD IC₅₀ (nM) 7F6 3 21E6 11

Due to differences in this assay from the previous iteration, the calculated IC50s are not directly comparable with those previously recorded. Despite this, the pairwise comparison between 7F6 and 21 E6 assayed on the same plate indicate that 21 E6 can still be considered a potent ICVD.

Example 5: Evaluation of the Intestinal Stability of Purified E. coli Recombinant ICVDs

Stability of purified E. coli recombinant anti-IL-6R ICVDs in human faecal extract ICVDs were digested in pooled human faecal extract for 6 h each as detailed in ‘the standard human faecal extract digestion assay’. The digested samples were then analysed as detailed in ‘the standard anti-IL-6R gp130 ELISA performed on samples previously exposed to proteolytic material’. Any protocol deviations are described below. These assays can be found above under the heading ‘Stability’.

Briefly, Maxisorp 96-well plates were coated overnight with 50 μl/well 0.5 μg/ml gp130 then blocked. ICVDs were serially diluted on ice and mixed 1:1 with 200 ng/ml recombinant human IL-6, then 1:1 with 20 ng/ml recombinant human sIL-6R and incubated for 10 mins before adding to the gp130-coated plates. Bound IL-6R was detected with 50 μl/well 0.250 μg/ml BAF227 and then 50 μl/well 1/1000 Extravidin-HRP and the level of neutralisation by the ICVD of IL-6R binding to IL-6/gp130 was determined.

The amount of ICVD remaining in the 0 time point and the 6 h digested samples was interpolated from standard curves using the Graphpad Prism software package. The ICVD concentration in the 6 h sample is expressed as a % of that in the 0 h sample to give ICVD survival. The results are shown in Table 5.1.

TABLE 5.1 Assay 1 - Stability of ICVDs after 6 hours' incubation in pooled human faecal extract Name 7F6 5G9 3C12 4D3 % survival 42 75 16 46

A separate assay on 21E6 was performed using the protocol as described for the 21E6 potency assays in the section “Inhibition of gp130/IL-6/IL-6R complex formation—potency in neutralising soluble human IL6R”. The results of the assay are shown in Table 5.2.

TABLE 5.2 Assay 2 - Stability of 21E6 after 6 hours' incubation in pooled human faecal extract Name 21E6 % survival 50.8 Stability of Purified E. coli Recombinant Anti-IL-6R ICVDs in Mouse Small Intestinal Supernatant

ICVDs were digested in mouse small intestinal supernatant for 1 h each as detailed in ‘the standard mouse small intestinal supernatant digestion assay’. The digested samples were then analysed as detailed in ‘the standard anti-IL-6R gp130 ELISA performed on samples previously exposed to proteolytic material’. Any protocol deviations are described below. These assays can be found above under the heading ‘Stability’.

Briefly, Maxisorp 96-well plates were coated overnight with 50 μl/well 0.5 μg/ml gp130 then blocked. ICVDs were serially diluted on ice and mixed 1:1 with 200 ng/ml recombinant human IL-6, then 1:1 with 20 ng/ml recombinant human sIL-6R and incubated for 10 mins before adding to the gp130-coated plates. Bound IL-6R was detected with 50 μl/well 0.250 μg/ml BAF227 and then 50 μl/well 1/1000 Extravidin-HRP and the level of neutralisation by the ICVD of IL-6R binding to IL-6/gp130 was determined.

The amount of ICVD remaining in the 0 time point and the 1 h digested samples was interpolated from standard curves using the Graphpad Prism software package. The ICVD concentration in the 1 h sample is expressed as a % of that in the 0 h sample to give ICVD survival. The results are shown in Table 5.3.

TABLE 5.3 Assay 1 - Stability of selected ICVDs after 1 hour incubation in mouse small intestinal supernatant Name 7F6 5G9 4D3 % survival 18 23 46

A separate assay on 7F6 and 21E6 was performed using the same protocol as described for 21E6 human faecal digestions. The results of the assay are shown in Table 5.4.

TABLE 5.4 Assay 2 - Stability of 21E6 after 1 hour incubation in mouse small intestinal supernatant Name 21E6 % survival 29.5

In summary, it was found that ICVDs 7F6, 5G9 and 21E6, which all share similar CDR3 sequences, are not only highly potent but also surprisingly stable to intestinal proteases.

Example 6: Overall Summary of Properties of Selected ICVDs

ICVDs have been identified with good potency in assays measuring neutralisation of human and cynomolgus monkey soluble IL-6R, membrane IL-6R and resistance to inactivation by intestinal proteases. These characteristics are summarised in Table 6.1, which incorporates data from both first and second rounds of assays.

TABLE 6.1 Potency and protease resistance characteristics of ICVDs % Survival 1 hour gp130 Binds mouse ELISA TF1 assay cyno small % Survival 6 EC₅₀ (nM) EC₅₀ (nM) monkey intestinal hours human Identifier (sIL-6R) (mIL-6R) sIL-6R supernatant faecal extract 3C12 1.89 44.6 Yes NT 16 4D3 0.66 26.0 Yes 46 46 5G9 0.16 0.9 Yes 23 75 7F6 0.11 0.5 Yes 18 42 21E6 0.3 11 NT 29.5 50.8 (NT = not tested)

It can be seen that ICVDs 7F6, 5G9 and 21E6, which all share similar CDR3 sequences, are highly potent and highly stable to intestinal proteases. 4D3, which also has a distinct CDR3 sequence to 7F6, 5G9 and 21E6, has relatively poorer potency. 3C12, which has a distinct CDR3 sequence to 7F6, 5G9 and 21E6, has relatively poorer potency and relatively poorer protease stability.

Example 7: Epitope Analysis

The selected ICVDs were assayed for their ability to cross-compete with tocilizumab Fab for binding to IL-6R.

Four Nunc Maxisorp 96-well ELISA plates were coated with 1 μg/ml mouse anti-human IgG Fab fragment (abcam ab1927) in PBS and incubated overnight at 4° C. Plates were washed in wash buffer (0.05% Tween in 1×PBS) using an automatic plate washer and blocked in block buffer (1% BSA in PBS) for 1 hour. Stocks of 40 ng/ml IL-6R, 8 nM TCZ (tocilizumab) Fab, and 400 nM ICVDs were prepared in block buffer. ICVD solutions were made up from a masterplate of 5 μM stock solutions.

Solutions of purified ICVDs were transferred to a Dilution Plate, where they were serially diluted threefold in 1% BSA in PBS. 60 μl each diluted sample was mixed with 60 μl TCZ Fab. 85 μl of this mixture was subsequently mixed with 85 μl IL-6R solution and incubated on a shaking platform for 1 hour. This resulted in final concentrations of: 100-0.138 nM ICVD, 2 nM TCZ Fab, and 20 ng/ml IL-6R. Block buffer was removed from ELISA plates and 50 μl each sample was added to each well in triplicate. Samples were incubated on the assay plates for 2 hours. Plates were washed as above, and 50 μl per well of 0.25 μg/ml BAF227 anti-IL-6R (R&D Systems) in blocking buffer was added to the plate and incubated for 1 hour. The plates were washed as above and incubated with 50 μl/well of a 1/1000 dilution of ExtrAvidin peroxidase (Sigma E2886) in blocking buffer for 1 hour. Plates were washed as above, and 100 μl TMB Microwell Substrate (KPL) was added. After 20 minutes, the reactions were stopped with 50 μl 0.5 M H₂SO₄ per well. OD₄₅₀ was measured using a BMG FluoStar Omega plate reader.

Analysis of Data:

OD₄₅₀ values for the highest concentration of ICVD (100 nM) were used to represent the maximum level of tocilizumab competition achievable by the ICVDs. The percentage competition with the tocilizumab Fab was calculated as a percentage of the maximum range of the assay, with this value calculated for a non-IL6R binding ICVD control (IRR) included in each masterplate subtracted:

${\% \mspace{14mu} {tocilizumab}\mspace{14mu} {competition}} = {\left( \frac{{{OD}\mspace{14mu} {NoVHH}} - {{OD}\mspace{14mu} {Sample}}}{{OD}_{NoVHH} - {OD}_{{IL}\; 6\; R\mspace{14mu} {only}}} \right) - {\% \mspace{14mu} {competition}\mspace{14mu} {of}\mspace{14mu} {IRR}}}$

Results

The ICVDs were sorted according to their ability to inhibit the binding of the tocilizumab Fab to IL-6R (% tocilizumab competition, as shown in Table 7.1.

TABLE 7.1 Calculated average OD450 values and % tocilizumab completion values ranked by % competition: % ICVD competition 20A11 102.78 (prior art comparative example) 3C12 81.72 7F6 73.64 5G9 33.36

It can be seen from this competition analysis that 7F6 and particularly 5G9 may be expected to bind to a different epitope to that of tocilizumab or 20A11.

Example 8: Mutations to the 5G9 and 7F6 Sequences and Impact on Potency Mutant 5G9 Sequences

Mutant 5G9 sequences were produced and tested for their potency in the standard gp130 ELISA. The standard gp130 ELISA has a final assay concentration of 10 ng/mL IL-6R and 50 ng/mL IL-6. The results are shown in Table 8.1 below.

TABLE 8.1 Potency of mutant 5G9-based sequences in the gp130 ELISA Name Mutations Mutation(s) location Potency 5G9 ‘Wild type’ — 0.09 ID-52V R105H F109H CDR3/CDR3 0.24 ID-53V F109H CDR3 0.30 ID-54V R105H CDR3 0.15 ID-55V K23H FR1 0.11 ID-56V F29S FR1 0.07 ID-57V F29I FR1 0.31 ID-58V F65V CDR2 0.36 ID-59V Y62D CDR2 0.11

IC50s were increased by approximately threefold in the F291 and F65V mutants, but all mutants nonetheless maintained high potency in this assay.

Some of these sequences were also tested for their potency in the TF-1 assay. The results are shown in Table 8.2 below.

TABLE 8.2 Potency of mutant 5G9-based sequences in the TF-1 assay Name Mutations Mutation(s) location Potency tocilizumab — — 0.69 5G9 ‘Wild type’ — 0.23 ID-52V R105H F109H CDR3/CDR3 14.20 ID-53V F109H CDR3 17.52 ID-54V R105H CDR3 0.31 ID-56V F29S FR1 0.33

The mutations were well tolerated in this assay.

Mutant 7F6 Sequences

Mutant 7F6 sequences were produced and tested for their potency in the standard gp130 ELISA. The results are shown in Table 8.3 below.

TABLE 8.3 Potency of mutant 7F6-based sequences in the gp130 ELISA Name Mutations Mutation(s) location Potency 7F6 ‘Wild type’ — 0.15 ID-3V R102H CDR3 0.16 ID-6V F106H CDR3 0.22 ID-40V R102H F106H CDR3/CDR3 0.21 ID-47V F106I CDR3 0.27 ID-49V F106T CDR3 0.31 ID-50V F106V CDR3 0.24

It can be seen that these substitutions in the FRs or CDRs of the 5G9 and 7F6 sequences can be tolerated without seriously impacting potency.

Example 9: Modification of Cis/Trans-Binding Profile of 509

Selectivity for antagonism of IL-6 trans-signalling over cis-signalling can be defined by inhibitory potency in the IL-6-sIL-6R-gp130 ELISA (surrogate for trans-signalling) compared to inhibitory potency in the membrane IL-6R dependent TF-1 cell assay (surrogate for cis-signalling).

It was noted that the introduction of the F109H mutation led to a marked shift in activity towards selective antagonism of IL-6 trans-signalling (see potency of ID-53V, incorporating F109H, compared to ‘Wild type’ 5G9, in the gp130 ELISA and the TF-1 assays in Example 8 above).

Accordingly, trans-selective versions of 5G9 (ID-122V, ID-123V) underwent further optimisation in parallel with the cis-trans potent versions of 5G9 (ID-112, ID-114) as detailed below.

Example 10: Optimisation of 5G9-Derived ICVDs for Yeast Production

Constructs were designed initially for production of ICVDs in E. coli, necessitating C-terminal Flag- and His-tags to aid purification of the recombinant proteins. 5G9-derived ICVDs were tested for production in yeast without an epitope tag and with an E1D amino acid substitution to reduce the potential for formation of N-terminal pyroglutamate and resultant heterogeneity of final product.

ICVDs ID-74V (5G9 but for E1D and R105H mutations) and ID-75V (5G9 but for E1D, F29S and R105H mutations) were cloned into the yeast integration vector pUR9013 and transformed into S. cerevisiae. Experiments were performed to confirm that the yeast-expressed ICVDs retained the characteristics of the earlier E. coli-produced ICVDs. Yeast-produced ICVDs maintained substantially the same potency as E. coli-produced ICVDs in the standard gp130 ELISA. Protease resistance was also very similar between the yeast- and E. coli-produced ICVDs.

Example 11: Further Optimisation of 5G9

A selection of further mutations were introduced into 5G9-derived ICVDs in combination with the mutations identified above and the resultant sequences produced in E. coli. These are shown in Table 11.1.

TABLE 11.1 Optimised variant 5G9-based ICVDs Construct Expression Antagonism Mutations relative to 5G9 C-terminus ID-112V E. coli Cis/Trans F29S; Q47G; K89R; R105H FLAG-6xHis ID-114V E. coli Cis/Trans F29S; V81L; N82Y; R105H FLAG-6xHis ID-122V E. coli Trans F29S; Q47G; K89R; FLAG-6xHis R105H; F109H ID-123V E. coli Trans F29S; Q47G; N82Y; K89R; FLAG-6xHis R105H; F109H ID-141V Yeast Trans E1D; F29S; Q47G; V81L; Wild type N82Y; K89R; R105H; F109H ID-142V Yeast Trans E1D; F29S; Q47G; N82Y; Wild type K89R; R105H; F109H ID-143V Yeast Cis/Trans E1D; F29S; Q47G; V81L; Wild type N82Y; K89R; R105H ID-144V Yeast Cis/Trans E1D; F29S; Q47G; N82Y; Wild type K89R; R105H

A ‘Wild type’ C-terminus denotes the natural ending of a llama ICVD domain sequence. 6×His-FLAG denotes the functionally inert affinity tag used for purification of E. coli-derived constructs. Labile residues are present between the His tag and the C terminus of the ICVD. Western blot analysis demonstrates that the His tag is completely cleaved from the ICVD before potency or stability analysis takes place.

It can be seen that ID-123V is equivalent to ID-142V except for ID-123V being produced in E. coli and therefore lacking the E1D mutation. ID-112V is equivalent to ID-144V except for (a) ID-112V lacking the N82Y mutation and (b) ID-112V being produced in E. coli and therefore lacking the E1D mutation.

Example 12: Potency of Optimised ICVDs Against Soluble and Membrane Human IL-6R in Various Assays

The inhibitory potency and efficacy (maximal inhibition) of the E. coli-produced 5G9 derivatives were confirmed in vitro in the gp130 ELISA, TF-1 cell and M1 cell functional assays measuring responses mediated via human IL-6R. The potency of the E-coli-produced 5G9 derivatives in these assays is shown in Tables 12.1-12.7. The gp130 assays follow the standard protocol with a final assay concentration of 10 ng/mL IL-6R and 50 ng/mL IL-6.

gp130 ELISA Assays

TABLE 12.1 Potency of 5G9 derivatives - gp130 ELISA assay - Experiment 1 Construct gp130 ELISA EC₅₀ nM ID-112V 0.1496 ID-114V 0.1375

TABLE 12.2 Potency of 5G9 derivatives - gp130 ELISA assay - Experiment 2 Construct gp130 ELISA EC₅₀ nM ID-122V 0.489 ID-123V 0.498 Tocilizumab 1.831 (prior art comparative example) 20A11 0.220 (prior art comparative example)

TF-1 Cell Assays

TABLE 12.4 Potency of 5G9 derivatives - TF-1 cell assay - Experiment 1 TF-1 cell assay EC₅₀ nM Construct (5 ng/ml IL-6) ID-112V 0.18 Tocilizumab 0.69 (prior art comparative example)

TABLE 12.5 Potency of 5G9 derivatives - TF-1 cell assay - Experiment 2 TF-1 cell assay EC₅₀ nM Construct (5 ng/ml IL-6) ID-123V 4.50 Tocilizumab 1.12 (prior art comparative example) 20A11 0.56 (prior art comparative example)

TABLE 12.6 Potency of 5G9 derivatives - TF-1 cell assay - Experiment 3 TF-1 cell assay EC₅₀ nM Construct (5 ng/ml IL-6) ID-114V 0.29

M1 Cell Assay

The M1 cell assay was developed to investigate the IL-6 trans-signalling neutralising potency of ICVDs specific for IL-6R. M1 cells respond to exogenous IL-6+IL-6R by inhibition of proliferation, by differentiation and eventually cell death, but this response can be negated by IL-6R specific ICVDs that prevent IL-6R-IL-6 binding.

The M1 assay measures the effect, in terms of cell behaviour, of ICVD blockade of IL-6 trans-signalling over a period of days. In assessment of ICVD trans selectivity, the TF-1 cell assay is used to measure cis IL-6 signalling; it is therefore appropriate when determining the degree of trans vs cis selectivity, to use data from another cellular assay, dependent on IL-6 trans signalling, such as the M1 cell assay, for comparison.

M1 cells from a rapidly growing stock culture were inoculated into 96-well assay plates in RPMI medium+3% FCS at 5×10⁴ cells/50 μl/well. IL-6, IL-6R and concentration ranges of ICVDs were all prepared in the same medium at 6× the final assay concentration (20 ng/ml IL-6R, 100 ng/ml IL-6). 75 μl of each ICVD dilution was mixed with the same volume of 120 ng/ml IL-6R and incubated for 1 hour at room temperature, before addition of 75 μl of 600 ng/ml IL-6 to each of these mixtures. Finally, 50 μl of each ICVD/IL-6R/IL-6 mixture was added to four replicate wells of M1 cells, and the plates incubated undisturbed for 5 days. Viable cell mass was then measured by Alamar blue addition (10 μl/well), further incubated for 5 hours, and finally 50 μl 3% SDS solution was added, and fluorescence measured at 620 nm. IL-6 only and IL-6+IL-6R only controls represent 0% and 100% of the IL-6+IL-6R stimulated response. The results of the M1 cell assay are shown in Table 12.7 below.

TABLE 12.7 Potency of 5G9 derivatives - M1 cell assay Construct M1 cellular assay EC₅₀ nM ID-112V 0.55 ID-114V 0.52 ID-122V 3.96 ID-123V 4.20 Tocilizumab 5.36 (prior art comparative example)

Cynomolgous Monkey IL-6R Competition Assay

It was found that ID-123V was active in the cynomolgus monkey IL-6R competition assay.

Summary of Potency Assays Performed on 5G9 Derivatives

In summary, it can be seen that these 5G9-derived ICVDs maintained high potency in the various assays performed.

Example 13: Evaluation of the Intestinal Stability of Optimised ICVDs Assessment in Mouse Small Intestinal Supernatant and Human Faecal Extract Assays

Various 5G9 variants and 20A11, a comparative anti-IL-6R polypeptide of the prior art, were tested for their survival in the standard mouse small intestinal supernatant and human faecal extract assays. Survival was measured by the gp130 plate ELISA as laid out above under ‘Stability’; ‘the standard Anti-IL-6R gp130 ELISA performed on samples previously exposed to proteolytic material’.

The standard mouse small intestinal supernatant assay was performed on these polypeptides with 4 hours' and 7 hours' digestion and the standard human faecal extract assay was performed on these polypeptides with 16 hours' digestion (using 250 ug/mL in 0.1% BSA assay stocks diluted to 20 ug/mL in protease testing solution). % survival was established for these polypeptides as follows in Table 13.1.

TABLE 13.1 Mouse small intestinal supernatant and human faecal extract stability of 5G9 variants Mouse Mouse small small Human intestinal intestinal faecal supernatant supernatant extract 4 h 7 h 16 h % ICVD Modifications % survival % survival survival 112V F29S Q47G K89R R105H 44 NT 74 114V F29S V81L N82Y R105H 82 NT 99 122V F29S, Q47G; K89R; R105H, NT 73 49 F109H 123V F29S Q47G; N82Y K89R; NT 85 95 R105H, F109H 142V E1D; F29S; Q47G; N82Y; NT 47 92 K89R; R105H; F109H 20A11 — NT 0 0 (prior art comparative example) NT = not tested

It is recalled that in the earlier assays detailed above, ‘wild type’ 5G9 demonstrated 23% survival after 1 hour's exposure to mouse small intestinal supernatant and 75% survival after 6 hours' exposure to human faecal supernatant.

It can be seen that these mutations generated highly protease resistant ICVDs. This was particularly evident in the trans-selective ICVD ID-123V, which showed extremely high survival in the presence of intestinal proteases (85% after 7 h in mouse small intestinal extract and 95% after 16 h in human faecal supernatant). In contrast, it can be seen that 20A11 is highly unstable in the presence of intestinal proteases.

In addition, a less stringent 7 hour mouse small intestinal supernatant assay and a less stringent 16 hour human faecal extract assay were performed on ID-123V, ID-122V and 20A11. In these less stringent assays, the assay stocks were made up to 118 ug/mL in 0.1% BSA instead of 250 ug/mL in 0.1% BSA in the standard assay. % survival was established for these variant ICVDs as follows in Table 13.2.

TABLE 13.2 Mouse small intestinal supernatant and human faecal extract stability of 5G9 variants ID-123V, ID-122V and prior art anti-IL6R polypeptide 20A11 After 16 h in After 7 h Human in Mouse Faecal SI extract Extract ICVD Modifications % survival % survival ID-123V F29S Q47G; N82Y K89R; 99 80 R105H, F109H ID-122V F29S, Q47G; K89R; 73 49 R105H, F109H 20A11 — 0 0 (prior art comparative example)

As noted in the previous standard stability assays, these less stringent assays further confirm that ID-123V and ID-122V are highly protease resistant ICVDs. Furthermore, it can be seen that 20A11 is highly unstable in the presence of intestinal proteases.

Resistance to Gastrointestinal Matrix Metalloproteases

Levels of activated matrix metalloproteases (MMPs) are increased in the inflamed mucosa of patients with intestinal bowel disease. These MMPs are able to digest native human IgG and therapeutic agents that contain a human IgG scaffold (Biancheri et al, 2015). In the case of the anti-TNFα therapy etanercept, this digestion causes a significant reduction in TNFα neutralising potency. To confirm that the optimised ICVDs described here are resistant to MMPs, ID-112V and ID-123V were incubated for 18 hours in the presence of activated recombinant human MMP3 and MMP12.

Following incubation, both ID-112V and ID-123V were fully potent at neutralising IL-6R, as measured using the IL-6-sIL-6R-gp130 functional ELISA. However, after the same incubation time, MMP3 and MMP12 digested full-length etanercept and tocilizumab (anti-IL-6R) to smaller fragments, as measured by Western blotting.

Mouse Digestive Transit and Survival

Results of the in vitro studies described above showed that optimised 5G9-derivatives were highly resistant to inactivation by proteases present in a supernatant extract prepared from mouse small intestinal contents. A subsequent study was performed to investigate the stability of trans-selective ID-123V during passage through the gastrointestinal system of the mouse. ID-123V was formulated with a further, unrelated ICVD in a milk and bicarbonate mixture to protect against denaturation at low pH and digestion by pepsin in the stomach. Following administration of the ICVDs to mice by oral gavage, concentrations of the ICVDs in stomach, small intestine, caecum and colon were determined at 3 hours post-dosing. In addition, the ICVD concentration in faecal pellets collected at hourly intervals was measured.

Four mice were tested. The transit and excretion of the ICVDs in faeces varied between the mice tested. In three of the mice, ID-123V was detected in faeces between 2 and 4 hours after dosing, suggesting that ID-123V can survive transit through the mouse GI tract.

Examining the concentrations of ID-123V in each compartment at 3 hours post-dose revealed ID-123V in the stomach and small intestine. It is expected therefore that the 5G9 derived ICVDs will have good stability in the human intestinal tract.

Example 14: Binding Affinity and Specificity for Human IL-6R Biacore Estimation of Optimised ICVD—IL-6R Binding Affinity

The binding kinetics of the trans-selective ID-123V (E. coli produced version of ID-142V) and the cis/trans potent ID-112V (E. coli produced version of ID-144V, but for ID-112V lacking N82Y relative to ID-144V) were compared against tocilizumab in a Biacore study. The ICVDs were fixed to the Biacore sensor plate and soluble human IL-6R was flowed over the plate to detect binding. Tocilizumab and the trans-selective ID-123V had similar Kds of 0.39 and 1.1 nM, respectively, while ID-112V had a lower Kd of 40 pM. The results indicate that both the trans-selective and cis/trans potent ICVDs demonstrate strong binding to the antigen.

Specificity of Optimised ICVDs

The trans-selective 5G9 derivative ID-123V was tested for selectivity against proteins related to human IL-6R. Human IL-11R and CNTFR (ciliary neurotrophic factor receptor) were found to be the most closely related human proteins to IL-6R identified using the NCBI BLASTp tool, both with a sequence identity with IL-6R of 29%. In ELISA assays, none of human IL-11R, CNTFR, or IL-6 interfered with IL-6R binding to ID-123V, while addition of human IL-6R shifted the curve dramatically. This indicated that binding of 5G9-derived ICVDs such as ID-123V to off-target molecules would be very unlikely in humans.

Example 15A: Efficacy in Ex Vivo IBD Models (ID-123V)

The possibility of testing the efficacy of anti-IL-6R ICVDs in an in vivo preclinical animal model of inflammatory bowel disease was precluded by the lack of cross-species reactivity towards murine IL-6R and the absence of a suitable non-human primate model of IBD. Although humanised (human immune cell engrafted) NOG mice are available commercially, it is as yet unclear whether established methods (e.g. DSS-administration) would be effective for the induction of inflammatory colitis in these animals. Since murine non-haematopoietic cells may be expected to produce and/or respond to IL-6, the potential effectiveness of human-specific IL-6R antibodies in a NOG mouse colitis model is uncertain.

Phosphorylation of Signalling Proteins

Infliximab is effective for the treatment of IBD. Infliximab is thought to act primarily by neutralising the biological activity of TNF leading to inhibition of the downstream pro-inflammatory effects of the cytokine. Activation of the many different cell types present in diseased tissue by TNF and secondary inflammatory mediators involves multiple receptor signalling pathways resulting in the phosphorylation of receptors, protein kinases and transcription factors. Experiments have shown that (i) patterns of protein phosphorylation are altered in IBD vs normal intestinal tissue and that (ii) patterns of phosphorylation are sensitive to inhibitors of specific pro-inflammatory mechanisms.

Due to the lack of a suitable preclinical in vivo animal model of IBD, an alternative human ex vivo IBD tissue model that reproduces the inflammatory bowel disease tissue environment was used (see Vossenkimper et al, 2014). A study was conducted to investigate the activity of the 5G9-derived ICVD ID-123V (containing the same mutations as the yeast-produced trans-selective ICVD ID-142V, but for E1D) on protein phosphorylation patterns in this human ex vivo model.

ID-123V and controls (ID-2A (an anti C. difficile toxin ICVD) and tocilizumab) were tested in ex vivo cultures for their effects, after 4 hours and 24 hours incubation, on levels of phosphoproteins present in inflamed biopsy tissue obtained from five patients with active IBD (4 hour incubation results in FIGS. 7-11, 24 hour incubation results in FIGS. 12-16).

Based on a visual assessment of the phosphoprotein signal patterns (summarised in Table 15.1), responses to the IL-6R antagonist ICVD ID123V and tocilizumab were noted in tissues from 3/5 IBD patients. In tissues from two of the IBD patients (UC2045 and CD2059, FIGS. 7, 9, 12 and 14), the patterns of inhibition obtained with ID-123V and tocilizumab were very similar with marked suppression of a majority of the phosphoprotein levels. The lack of (or more restricted) responses to both ID-123V and tocilizumab in tissues from two other IBD cases (CD2052 and CD2061, FIGS. 8, 10, 13 and 15) may have reflected differences in IL-6 dependent disease activity or differences in the cellularity of the biopsies taken from these patients.

TABLE 15.1 Responses to anti-IL-6R agents Effect of anti-IL-6R agent compared with negative Control Effects on phospho-protein Effects on phospho-protein Patient # Anti-IL-6R signals at 4 h signals at 24 h UC2045 ID-123V Substantial inhibition - most Substantial inhibition - most markers markers Tocilizumab Substantial inhibition - most Substantial inhibition - most markers markers CD2052 ID-123V No inhibition - stimulation most Low level stimulation most markers markers Tocilizumab Partial inhibition - few markers Partial inhibition - most markers CD2059 ID-123V Substantial/complete inhibition - Substantial/complete inhibition - most markers most markers Tocilizumab Substantial/complete inhibition - Substantial inhibition - most most markers markers CD2061 ID-123V Partial inhibition of a few markers Partial inhibition of a few markers Tocilizumab Partial inhibition of most markers Partial inhibition of a few markers UC2075 ID-123V Substantial inhibition - some Substantial inhibition - >50% markers markers Tocilizumab Some inhibition - a few markers Some inhibition - a few markers

Overall, it can be seen that ID-123V produced a response that was highly similar to the clinical anti-IL-6R monoclonal antibody tocilizumab in this complex disease model system.

Production of Proinflammatory and Anti-Inflammatory Cytokines

A further study was conducted to investigate the activity of ID-123V in a human ex vivo model, reproducing the inflammatory bowel disease tissue environment.

IL-6 is important for the activation of inflammatory processes that contribute to the pathology and chronicity of IBD. Many of these effects rely on the regulation of complex cytokine networks that in turn control the different cellular processes involved in inflammation and immune-regulation.

ID-123V, negative control (ID-2A, anti C. difficile toxin ICVD) and the monoclonal anti-IL-6R antibody tocilizumab were tested for their effects on spontaneous production of proinflammatory and anti-inflammatory cytokines over a period of 24 h.

Aims of the study were to (i) determine whether the IL-6R-antagonist activity of ID-123V can be demonstrated in ex vivo cultures of IBD tissue based on changes in the levels of tissue cytokine production (ii) to compare effects of ID-123V with the clinically effective anti-IL-6R mAb tocilizumab.

Mucosal biopsies taken from active IBD cases with inflamed mucosa were cultured (one biopsy per well) in 24-well plates (VWR International) in 300 μl serum-free HL-1 medium (Cambrex BioScience) supplemented with glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 50 mg/mL gentamicin and cultured at 37° C., 5% CO₂. Biopsies were cultured for 24 h with the addition of the following agents; tocilizumab, ID-123V or ID-2A each at a final concentration of 70 nM. Supernatants collected at the end of the experiment (24 h) were snap-frozen and stored at −70° C. Samples of the culture supernatants were analysed for levels of IL-6, IL-8 and IL-10. The results are shown in FIGS. 17-19.

It can be seen that ID-123V inhibited spontaneous production of IL-6 and IL-8 from inflamed IBD tissues and stimulated production of IL-10 by biopsies from two UC patients. Overall, ID-123V demonstrated effects which were similar to those of tocilizumab.

Example 15B: Efficacy in Ex Vivo IBD Models (ID-142V)

Similar studies to those carried out in Example 15A were performed in respect of ID-142V.

Investigation of the Inhibitory Effects of ID-142V on the Phosphorylation of Signalling Proteins in Ex Vivo Cultured IBD Tissue IBD Tissue

Endoscopic colonic mucosal biopsies were obtained from patients with active Inflammatory Bowel Disease (IBD). Patient characteristics are listed in Table 15.2.

TABLE 15.2 Details of IBD Patients, Disease Presentation and Medication Sex; birth Patient # Presentation/Biopsy Medication year CD2241 /colon Azathioprine M; 1976 CD2244 /colon Azathioprine, Budesonide M; 1982 CD2250 /colon No meds; (on Humira 1 M; 1992 year ago) CD2256 /terminal ileum No meds M; 1985 UD2245 mild pancolitis Mesalazine oral and topical M; 1955 UD2249 mayo 1-2 colitis No meds F; 1985 left-sided

Organ Culture

Mucosal biopsies taken from active IBD cases (CD & UC) with inflamed mucosa were cultured (one biopsy per well) in 24-well plates (VWR International, Lutterworth, UK) in 300 μl serum-free HL-1 medium (Cambrex BioScience, Wokingham, UK) supplemented with glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin, 50 ug/mL gentamicin and cultured at 37° C., 5% CO₂. Biopsies were cultured for 24 h with the addition of ID-142V at a final concentration of 250 nM or ID-2A 500 nM (ID-2A is a negative control anti C. difficile toxin ICVD). Supernatants and tissue samples collected at the end of the experiment were snap-frozen and stored at −70° C.

Signalling Arrays and Data Analysis

For the analysis of phospho-protein content the IBD tissue samples were thawed, lysed in RIPA buffer (Sigma-Aldrich, St. Louis, Mo.) supplemented with phosphatase inhibitor cocktail 2 (Sigma-Aldrich) and protease inhibitor cocktail (Sigma-Aldrich), both at 1%. Protein concentrations of the lysates were determined by the Bio-Rad protein assay (Bio-Rad Laboratories, Hemel Hempstead, UK) and samples diluted to 1.0 mg/ml in Array Diluent Buffer.

-   1. The phosphorylation status of receptor tyrosine kinases and     signalling molecules was determined by using PathScan RTK signalling     arrays (Cell Signalling Technology, Danvers, Mass.). The array kit     allows for the simultaneous detection of 28 receptor tyrosine     kinases and 11 important signalling nodes, when phosphorylated at     tyrosine or other residues. -   2. A total of 150 ug of protein from each biopsy tissue lysate was     probed onto each array. For each CD and UC patient, the lysates     prepared from the set of antibody-treated biopsies (ID-2A and     ID-142V) were analysed on the same slide. -   3. The chemiluminescent signals of all arrays were detected on X-ray     films, and the pixel intensities were measured using ImageJ     software. -   4. For each antibody treatment (ID-2A or ID-142V), the     phosphoprotein signals obtained from the four different patient     biopsies (n=4 CD lysates) were used to calculate mean+/−SD pixel     intensity values that are shown in FIGS. 20-22 (shown in     ‘AU’—arbitrary units). The intensity values for different     phosphoproteins are displayed in each figure. -   5. To account for differences between patients the pixel array data     obtained for the experimental treatment were normalised against the     pixel values obtained for the corresponding patient and ID-2A     treatment control. Percentage inhibition of control values were     calculated and presented in FIGS. 23-25. Values for the ID-142V     treatment were derived from the analysis of lysates from four     different CD patients. -   6. For comparison, the treatment was repeated using inflamed biopsy     tissue from two patients with ulcerative colitis. For each antibody     treatment (ID-2A or ID-142V) the phosphoprotein pixel intensity     values obtained from the two different patient biopsies (n=2 UC     lysates) were used to calculate mean+/−SD pixel intensity values     that are shown in FIGS. 26-28.

Results

When compared with the ID-2A controls, the pixel intensities measured for the Crohn's biopsies treated with ID-142V showed that ID-142V inhibited the phosphorylation levels of all the signalling proteins on the array (see FIGS. 20-22). When values for the experimental treated samples were normalised to the respective ID-2A Crohn's tissue control (FIGS. 23-25), the average percentage inhibition (n=4 CD patients) measured for all phosphoproteins on the array following ID-142V treatment was 59% (range 43% to 100%). Many of these proteins including CSF-1R, Tyro-3, AxI, Akt, ZAP-70, Lck, Stat1 and Stat3 are known to have functions involved in the regulation of inflammatory cells including macrophages and T cells that contribute to the immunopathology of Crohn's disease. The changes in phosphorylation are therefore consistent with the established anti-inflammatory effects of known IL-6R-neutralising antibodies.

The pixel intensity values for individual phosphoproteins and the pattern of intensities on the arrays found in the control ID-2A-treated UC biopsy tissue samples (average values for n=2 UC patients) were generally quite similar to those detected in the Crohn's tissue (FIGS. 26-28). However, the levels of phosphorylation of EphB4, Tyro3, AxI and VEGFR2 appeared to be higher while phosphorylation of Ret was lower in UC compared with CD tissue.

Investigation of the Inhibitory Effects of ID-142V on the Production of Cytokines in Ex Vivo Cultures of IBD Tissue IBD Tissue

Endoscopic colonic mucosal biopsies were obtained from patients with active Inflammatory Bowel Disease (IBD). Patient characteristics are listed in Table 15.3.

TABLE 15.3 Details of IBD Patients, Disease Presentation and Medication Patient # Presentation/Biopsy Medication M/F CD2241 /colon Azathioprine M; 1976 CD2244 /colon Azathioprine, Budesonide M; 1982 CD2250 /colon No meds; (on Humira 1 M; 1992 year ago) CD2254 /ileum Azathioprine, prednisolone F; 1958 CD2256 /terminal ileum No meds M; 1985 CD2259 /colon Azathioprine M; 1975

Organ Culture

Mucosal biopsies (two biopsies per patient) were taken from six patients with active Crohn's disease. The inflamed mucosa biopsies were cultured (one biopsy per well) in 24-well plates (VWR International, Lutterworth, UK) in 300 μl serum-free HL-1 medium (Cambrex BioScience, Wokingham, UK) supplemented with glutamine, 100 μg/ml penicillin, 100 μg/ml streptomycin, 50 ug/mL gentamicin and cultured at 37° C., 5% CO₂. The biopsies (×2) from each patient were cultured for 24 h with the addition of ID-142V at a final concentration of 250 nM or ID-2A (ID-2A is a negative control anti C. difficile toxin ICVD) at a concentration of 500 nM. Supernatants and tissue samples collected at the end of the experiment were snap-frozen and stored at −70° C.

Multiplexed Cytokine Assays

The 24 h culture supernatants recovered from each of the two ICVD-treated biopsies (ID-2A and ID-142V) were analysed for each of the six CD patients. The frozen culture supernatants were thawed and analysed for levels of IFN-gamma, TNF-alpha, IL-10, IL-17A, IL-1-beta, IL-6 and IL-8 using multiplexed cytokine assay kits and R&D Systems MagPix technology.

-   -   1. Samples were diluted 1:2 in reagent diluent.     -   2. Standards (custom mix by R&D Systems) were re-suspended and         1:3 serial dilutions were prepared.     -   3. The pre-mixed micro-particle cocktail (custom mix by R&D) was         prepared with reagent diluent.     -   4. Additions were made of 50 ul/well of standards and samples;         both in duplicates, plus 50 ul of micro-particle cocktail/well.     -   5. The plate was sealed and incubated for 2 h at RT on a shaker     -   6. The plate was then attached to a magnet and washed using a         multichannel pipette 3× with wash buffer.     -   7. 50 ul of biotin Ab cocktail (custom mix by R&D) was added per         well. The plate was resealed and incubated on a shaker for 1 h         at RT.     -   8. The wash step was repeated wash 3× using the magnet.     -   9. 50 ul of Streptavidin-PE mix was added/well and the plate         incubated for a further 30 min at RT on a shaker.     -   10. After washing 3×, the microparticles were resuspended by         adding 100 ul wash buffer/well, followed by incubation on a         shaker for 2 minutes.     -   11. The plate was read using a Luminex analyser. The MagPix         machine used was calibrated with R&D's calibration kit before         each run.

Assay data obtained for cytokine standards were used to generate standard curves; cytokine concentrations present in the culture supernatants were then calculated from the respective standard curves.

Data Analysis

The 24 h culture supernatants recovered from the ICVD-treated biopsies (ID-2A and ID-142V) were analysed for each of the six CD patients. The cytokine concentrations were determined and the mean values (Mean+/−Standard Deviation) were calculated using the results obtained from the set of six donors. Results are summarised in FIGS. 29-31.

Differences in the levels of inflammation and/or cellular involvement in biopsies taken from different patients could potentially result in different levels of spontaneous cytokine production between patients. To allow treatment effects to be more clearly identified the cytokine concentration value obtained for the experimental treatment was normalised to the value obtained for the corresponding patient ID-2A treatment control. The normalised % control values obtained for each cytokine (IFN-gamma, TNF-alpha, IL-10, IL-17A, IL-1-beta, IL-6 and IL-8) measured for each patient were then combined and the mean values (Mean+/−SD; n=6 CD patients) calculated. Normalised (% control) cytokine values obtained for the treatment (ID-142V/ID-2A) are presented in FIG. 32.

Results

When compared with the production levels of cytokines measured for the control ID-2A treated tissue, ID-142V treatment resulted in some inhibition of each of the six pro-inflammatory cytokines (FIGS. 29-31). The spontaneous production of IL-17 and TNF-alpha were inhibited by ID-142V most strongly. The control-normalised cytokine data presented in FIG. 32 also show that ID-142V treatment was inhibitory (FIG. 32).

In contrast to the inhibitory effects of ID-142V on production of the pro-inflammatory cytokines, treatment with ID-142V increased production of the anti-inflammatory cytokine IL-10 relative to control ID-2A (FIG. 29).

Example 16: Local Delivery to the Intestinal Tract and Access to Lamina Propria Following Oral Administration

Penetration of ID-123V into the lamina propria of mouse colons was assessed following oral dosing of dextran sulphate sodium (DSS).

DSS colitis was induced in mice using a standard protocol. 2% dextran suphate (MP biomedical) was administered in drinking water for 7 days, after which mice were kept for a further 3 days to allow peak development of disease.

On the day of peak disease, all mice received an initial pre-dose of bicarbonate+milk (vehicle) by oral gavage, followed 10 minutes later by further gavage of vehicle only, or vehicle+140 μg of ID-123V. At 1, 3, 5 and 7 hours post-dosing, pairs of animals were culled, the colons removed, and then colon segments were cryo-embedded for immunocytochemical analyses. Briefly, colon segments were cut and embedded in optimal cutting temperature compound (OCT) and snap frozen and stored at −80° C. until use. 6 um sections were cut and were fixed in ice-cold acetone for 90 seconds. The sections were air dried and stored at −20 degrees C. until assayed. Two serial sections for each mouse were used to stain for each antibody set.

Colon sections were incubated with a rabbit anti-ICVD polyclonal antibody, followed by an Alexa 594-linked goat anti-rabbit antibody and Hoechst 33342 nucleic acid stain. ID-123V associated immunofluorescence within the colon at different times after dosing is shown in FIG. 33 (blue=cell nuclei, red=ID-123V staining).

After 1 hour post-dosing, ID-123V was observed throughout the lamina propria and in the muscularis mucosa and was also detected at 3 and 5 hours. Interestingly, ID-123V had accessed the muscularis mucosa in colons of mice with severe disease at 1 and 3 hours, but penetration was largely confined to the upper epithelium in mice with mild disease, with only very low levels detectable in the muscularis mucosa. The data show that the anti-IL-6R ICVD ID-123V is able to penetrate the colon epithelium and submucosa of colitis mice following oral dosing.

Example 17: Sequence Analysis

Using high throughput sequencing, the heavy chain repertoire of DNA sequences obtained in Example 1 was established and collated in a database. Sequence analyses were performed on this database.

To statistically identify the germline amino acid sequences which gave rise to 5G9 and 7F6, an alignment of each sequence with reference V and J sequences was performed using Mafft (Katoh and Standley, 2013), and then V and J phylogenetic trees were generated using maximum parsimony methods. This gave the relationships between the germline sequences and 5G9 and 7F6, where the statistical support can be determined by bootstrapping.

Bootstrapping is a computational technique for assessing the accuracy of a statistical estimate, and, when applied to a phylogenetic tree, it represents the confidence of a branch division. Bootstrapping re-samples the positions of the sequence of interest, rebuilds the tree, and tests whether the same node/tree structure is recovered. This is repeated multiple times (×1000 in this analysis), and the percentage at each node represents the support for that branch.

The phylogenetic trees suggested that 5G9 and 7F6 are most closely related to the VHH-V gene Vo (confidence of 96%). For the VHH-J amino acid region, 5G9 and 7F6 are most closely related to VHHJ6 family (confidence of 97%).

Further sequence analysis was carried out to determine how many ICVD polynucleotide sequences from the database originated from the Vo germline (or related V genes) and possess the same V mutations as 21E6, 5G9 or 7F6, with and without the same CDR3.

Methods Calculating Germline Equivalent Sequences

All sequences in the database were aligned by the BLAST algorithm to the reference germline sequences, and hybrid sequences were generated where regions aligned to the reference germline sequences were combined with non-template regions of the ICVD sequences (Table 17.1). Note that 5G9 has a 3 amino acid insertion in framework 1 region compared to VHH Vo germline sequence

TABLE 17.1 Germline equivalents of ICVD sequences ID VHHR sequence Germline equivalent* 7F6 SGGGLVQAGGSTRLTCLASGSIS sggglvqaggslrlscaasg SINVIGWYRQAPGKQRELVAMIG sifsinamgwyrqapgkqre RGEGANYGDFAKGRFTISRDNSK lvaaltsggstnyadsvkgr NTVYLQMNSLKPEDTAVYYCYAD ftisrdnakntvylqmnslk YEDRDSPFNGSWGQG pedtavyycnaDYEDRDSPF (SEQ ID NO: 91) Ngswgqg (SEQ ID NO: 94) 5G9 SGGGLVQAGGSTRLTCKASGSIF sggglvqaggslrlscaasg NINSINVMAWYRQAPGKQRELVA sifNINsinamgwyrqapgk IIGKGGGTNYADFVKGRFTISRD qrelvaaitsggstnyadsv AAKNTVNLQMNSLKPEDTAVYY kgrftisrdnakntvylqmn ACYDYEDRDSPFNGSWGQG slkpedtavyycnaDYEDRD (SEQ ID NO: 92) SPFNgswgqg (SEQ ID NO: 95) 21E6 SGGGLVLAGGSTRLTCLASGSIS glvqaggslrlscaasgsif SINVIGWYRQAPGKQRELVAMIG sinamgwyrqapgkqrelva RGEGANYGDFAKGRFTISRDNSK aitsggstnyadsvkgrfti NTVYLQMNSLKPEDTAVYYCYAD srdnakntvylqmnslkped YEDRDSPLNGSWGQG tavyycnaDYEDRDSPLNgs (SEQ ID NO: 93) wgqg (SEQ ID NO: 96) *Lower-case letters indicate regions that were germline encoded.

Particular conserved differences from the germline equivalent sequence (including non-template regions) were identified. These differences are provided in Table 17.2.

TABLE 17.2 Conserved sites in the ICVD sequences which differ from germline (position numbering according to Kabat) Amino acid Conserved amino Germline amino position acid acid(s) 18 T L 21 T S 33 V* A 52 G* T 56 G* S 62 F S 93 Y* N

It is believed that the amino acids at the positions marked with an asterisk above are particularly important for maintaining optimal potency.

CLAUSES

A set of clauses defining the invention and its preferred aspects is as follows:

-   1. A polypeptide comprising an immunoglobulin chain variable domain     which binds to IL-6R, wherein the immunoglobulin chain variable     domain comprises three complementarity determining regions     (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3     comprises a sequence sharing 65% or greater sequence identity with     SEQ ID NO: 3. -   2. The polypeptide according to clause 1, wherein CDR3 comprises a     sequence sharing 80% or greater sequence identity with SEQ ID NO: 3. -   3. The polypeptide according to clause 1, wherein the sequence of     CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 48     to 53 or SEQ ID NO: 55 to SEQ ID NO: 58. -   4. The polypeptide according to any one of clauses 1 to 3, wherein     CDR1 comprises a sequence sharing 50% or greater sequence identity     with SEQ ID NO: 1. -   5. The polypeptide according to any one of clauses 1 to 3, wherein     CDR1 comprises a sequence sharing 40% or greater sequence identity     with SEQ ID NO: 8. -   6. The polypeptide according to any one of clauses 1 to 5, wherein     CDR2 comprises a sequence sharing 50% or greater sequence identity     with SEQ ID NO: 2. -   7. The polypeptide according to any one of clauses 1 to 5, wherein     CDR2 comprises a sequence sharing 50% or greater sequence identity     with SEQ ID NO: 9. -   8. The polypeptide according to clause 1 which comprises any one of     SEQ ID NOs: 15-39, 43 or 44. -   9. The polypeptide according to any one of clauses 1 to 8, wherein     the polypeptide is an antibody. -   10. The polypeptide according to any one of clauses 1 to 8, wherein     the polypeptide is an antibody fragment. -   11. The polypeptide according to any one of clauses 1 to 10, which     is substantially resistant to one or more proteases. -   12. The polypeptide according to any one of clauses 1 to 11 for use     as a medicament. -   13. The polypeptide according to clause 12 for use in the treatment     of an autoimmune and/or inflammatory disease. -   14. The polypeptide for use according to either clause 12 or 13,     which is administered orally. -   15. A polynucleotide encoding the polypeptide according to any one     of clauses 1 to 14, especially wherein the polynucleotide comprises     or consists of a sequence sharing 70% or greater sequence identity     with any one of SEQ ID NOs: 59 to 80, more especially wherein the     polynucleotide comprises or consists of any one of SEQ ID NOs: 59 to     80.

MISCELLANEOUS

All references referred to in this application, including patent and patent applications, are incorporated herein by reference to the fullest extent possible.

Throughout the specification and the claims which follow, unless the context requires otherwise, the word ‘comprise’, and variations such as ‘comprises’ and ‘comprising’, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps.

The application of which this description and claims forms part may be used as a basis for priority in respect of any subsequent application. The claims of such subsequent application may be directed to any feature or combination of features described herein. They may take the form of product, composition, process, or use claims and may include, by way of example and without limitation, the following claims.

SEQUENCES FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 >5G9 (SEQ ID NO: 15) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCKASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-52V (5G9 + F109H, R105H) (SEQ ID NO: 16) EVQLVESGGGLVQAGGSTRL INVMA WYRWAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPHNAS WGQGTQVTVSS TCKASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-53V (5G9 + F109H) (SEQ ID NO: 17) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPHNAS WGQGTQVTVSS TCKASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-54V (5G9 + R105H) (SEQ ID NO: 18) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-55V (5G9 + K23H) (SEQ ID NO: 19) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCHASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-56V (5G9 + F29S) (SEQ ID NO: 20) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-57V (5G9 + F29I) (SEQ ID NO: 21) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCKASGSIININS NYADFVKG MNSLKPEDTAVYYCYA >ID-58V (5G9 + F65V) (SEQ ID NO: 22) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCKASGSIFNINS NYADVVKG MNSLKPEDTAVYYCYA >ID-59V (5G9 + Y62D) (SEQ ID NO: 23) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDRDSPFNAS WGQGTQVTVSS TCKASGSIFNINS NDADFVKG MNSLKPEDTAVYYCYA >ID-112V (5G9 + F29S, Q47G, K89R, R105H) (SEQ ID NO: 24) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-114V (5G9 + F29S, V81L, N82Y, R105H) (SEQ ID NO: 25) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTLYLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-122V (5G9 + F29S, Q47G, K89R, R105H, F109H) (SEQ ID NO: 26) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPHNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-123V (5G9 + F29S, Q47G, N82Y, K89R, R105H, F109H) (SEQ ID NO: 27) EVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTVYLQ DYEDHDSPHNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-141V (5G9 + E1D, F29S, Q47G, V81L, N82Y, K89R, R105H, F109H) (SEQ ID NO: 28) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTLYLQ DYEDHDSPHNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-142V (5G9 + E1D, F29S, Q47G, N82Y, K89R, R105H, F109H) (SEQ ID NO: 29) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTVYLQ DYEDHDSPHNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-143V (5G9 + E1D, F29S, Q47G, V81L, N82Y, K89R, R105H) (SEQ ID NO: 30) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTLYLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >ID-144V (5G9 + E1D, F29S, Q47G, N82Y, K89R, R105H) (SEQ ID NO: 31) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKGRELVA IIGKGGGT RFTISRDAAKNTVYLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLRPEDTAVYYCYA >7F6 (SEQ ID NO: 32) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPFNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-3V (7F6 + R102H) (SEQ ID NO: 33) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKORELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDHDSPHNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-6V (7F6 + F106H) (SEQ ID NO: 34) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPHNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-40V (7F6 + R102H, F106H) (SEQ ID NO: 35) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDHDSPHNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-47V (7F6 + F106I) (SEQ ID NO: 36) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPINGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-49V (7F6 + F106T) (SEQ ID NO: 37) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPTNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >ID-50V (7F6 + F106V) (SEQ ID NO: 38) EVQLVESGGGLVQAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPVNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >21E6 (SEQ ID NO: 39) EVQLVESGGGLVLAGGSTRL INVIG WYRQAPGKQRELVA MIGRGEGA RFTISRDNSKNTVYLQ DYEDRDSPLNGS WGQGTQVTVSS TCLASGSISS NYGDFAKG MNSLKPEDTAVYYCYA >4D3 (SEQ ID NO: 40) EVQLVESGGGSVQAGESLTL QNAMG WFRQAAGKRRESVA RISSSGNV RFTMSRDNAKKTVYLQ YSMSGELAAP WGQGTQVTVSS SCVASISTFS GYTDAVKG MNSLKPEDTAVYYCNA >3C12 (SEQ ID NO: 41) EVQLVESGGGLAQLGGSLRL SNTAG WFRQAPGKQEEWVA GISIGGMP RFTISRDNAKNTVYLQ GGTEYDY WGQGTQVTVSS SCVASGNIFS AYADSVKG MNSLKPEDTAVYYCAT >20A11 (SEQ ID NO: 42) EVQLVESGGGLVQPGGSLEL INVMA WYRQAPGKGRELVA GIISGGST RFTISRDNAKNTLYLQ ITTESDYDLGRR WGQGTLVTVSS SCAASGSVFK SYADSVKG MNSLREEDTAVYYCAF Y >ID-74V (5G9 + E1D, R105H) (SEQ ID NO: 43) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSIFNINS NYADFVKG MNSLKPEDTAVYYCYA >ID-75V (5G9 + E1D, F29S, R105H) (SEQ ID NO: 44) DVQLVESGGGLVQAGGSTRL INVMA WYRQAPGKQRELVA IIGKGGGT RFTISRDAAKNTVNLQ DYEDHDSPFNAS WGQGTQVTVSS TCKASGSISNINS NYADFVKG MNSLKPEDTAVYYCYA

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1. A polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence having no more than 3 additions, substitutions and deletions compared to SEQ ID NO:
 3. 2. A polypeptide comprising an immunoglobulin chain variable domain which binds to IL-6R, wherein the immunoglobulin chain variable domain comprises three complementarity determining regions (CDR1-CDR3) and four framework regions (FR1-FR4), wherein CDR3 comprises a sequence having no more than 6 additions, substitutions and deletions compared to SEQ ID NO: 3 and wherein the immunoglobulin chain variable domain comprises one or more amino acids selected from V33, G52, G56 and Y93, and optionally one or more amino acids selected from T18, T21 and F62, according to Kabat numbering.
 3. The polypeptide according to claim 1, wherein CDR3 comprises a sequence having no more than 2 additions, substitutions and deletions compared to SEQ ID NO:
 3. 4. The polypeptide according to claim 1, wherein the sequence of CDR3 comprises any one of SEQ ID NO: 3, SEQ ID NO: 14, SEQ ID NO: 48 to 53 or SEQ ID NO: 55 to
 58. 5. The polypeptide according to claim 1, wherein CDR1 comprises a sequence having no more than 2 additions, substitutions and deletions compared to SEQ ID NO:
 1. 6. The polypeptide according to claim 5, wherein CDR1 comprises SEQ ID NO 1 or SEQ ID NO:
 8. 7. The polypeptide according to claim 1, wherein CDR2 comprises a sequence having no more than 8 additions, substitutions and deletions compared to SEQ ID NO:
 2. 8. The polypeptide according to claim 7, wherein CDR2 comprises any one of SEQ ID NOs: 2, SEQ ID NO: 9, SEQ ID NO: 46 or SEQ ID NO:
 47. 9. The polypeptide according to claim 1 which comprises any one of SEQ ID NOs: 15-39, 43 or
 44. 10. The polypeptide according to claim 1, wherein the polypeptide is an antibody.
 11. The polypeptide according to claim 1, wherein the polypeptide is an immunoglobulin chain variable domain.
 12. The polypeptide according to claim 11, wherein the polypeptide is selected from the list consisting of: a VHH, a VH, a VL, a V-NAR, a Fab fragment and a F(ab′)2 fragment.
 13. The polypeptide according to claim 12, wherein the polypeptide is a VHH.
 14. The polypeptide according to claim 1, wherein the polypeptide binds to TNF-alpha with a Kd of less than 10⁻⁶ M.
 15. The polypeptide according to claim 1, which is substantially resistant to one or more proteases present in the stomach or the small or large intestine.
 16. The polypeptide according to claim 1, wherein the polypeptide or construct retains 10% or more of the original potency of the polypeptide after at least 16 hours' exposure to human faecal extract in the standard human faecal extract assay.
 17. A pharmaceutical composition comprising the polypeptide according to claim 1 and one or more pharmaceutically acceptable diluents or carriers.
 18. The pharmaceutical composition according to claim 17 wherein the composition is presented in enterically coated form.
 19. A method of treating autoimmune and/or inflammatory disease comprising administering to a person in need thereof a therapeutically effective amount of the polypeptide according to claim
 1. 20. The method of treating autoimmune disease according to claim 19 wherein the polypeptide is administered orally. 